Myo-inositol oxygenases

ABSTRACT

The invention provides methods and materials related to the production of organic products such as glucuronic acid, ascorbic acid, and glucaric acid. Specifically, the invention provides cells, methods for culturing cells, isolated nucleic acid molecules, and methods and materials for producing various organic products such as glucuronic acid, ascorbic acid, and glucaric acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/472,317, filed Apr. 2, 2004, which is a U.S. national stageapplication under 35 U.S.C. §371 that claims the benefit ofPCT/US02/08404, filed Mar. 19, 2002, which claims the benefit of U.S.Provisional Application No. 60/277,148, filed Mar. 19, 2001.

BACKGROUND

1. Technical Field

The invention relates to methods and materials involved in producingorganic compounds such as organic acids.

2. Background Information

Ascorbic acid (vitamin C) has many important nutritional uses. In fact,ascorbic acid is an essential nutrient to humans, and must be obtainedfrom diet to prevent vitamin C deficiencies such as scurvy. In addition,some medical practitioners claim that ascorbic acid has the potential toprevent and treat the common cold, flu, and cancer. Thus, dietsupplements containing ascorbic acid are widely used.

Ascorbic acid also has many important industrial uses. For example,ascorbic acid can be used in meat processing, nutritional supplements,and animal foods. In fact, several industrial manufactures can produce10,000 metric tons annually of ascorbic acid and related ascorbic acidcompounds such as calcium ascorbate and sodium ascorbate.

The “Reichstein” method is a commonly used method for producing ascorbicacid from D-glucose or a D-glucose precursor such as corn syrup. Thismethod involves six discrete chemical steps as well as a fermentationstep. For example, one of the chemical steps involves converting2-keto-L-gulonic acid into ascorbic acid by treating the2-keto-L-gulonic acid with acid at a temperature greater than 60° C.

Several other manufacturing processes containing at least one chemicalstep are also used to produce ascorbic acid. Specifically, ascorbic acidhas been produced using methods that chemically convert D-glucose intoL-sorbitol prior to a fermentation step, methods that chemically convert2-keto-L-gulonic acid into ascorbic acid after a fermentation step, andmethods that chemically convert D-glucose into L-sorbitol prior to afermentation step in addition to chemically converting 2-keto-L-gulonicacid into ascorbic acid after a fermentation step.

SUMMARY

The present invention relates generally to methods and materials forproducing organic compounds such as myo-inositol, glucuronic acid,glucaric acid, and ascorbic acid. Specifically, the invention providescells (e.g., bacterial, fungal, and insect cells), methods for culturingcells, isolated nucleic acid molecules, and methods and materials forproducing various organic compounds. The invention is based on thediscovery that cells can be genetically manipulated such that they havethe ability to produce a desired organic product. For example, the cellsprovided herein can produce ascorbic acid. It will be understood thatthe terms “ascorbate,” “ascorbic acid,” “L-ascorbate,” “L-ascorbicacid,” and “vitamin C” can be used interchangeably to refer toL-ascorbic acid. It also will be understood that the term “glucaricacid” as used herein refers to glucaric acid, glucaro-1,4-lactone, andglucaro-6,3-lactone since these three compounds freely interconvert whenin solution.

The invention also is based on the discovery of efficient metabolicpathways that utilize glucose and/or phytic acid to produce ascorbicacid. Specifically, ascorbic acid can be produced from glucose and/orphytic acid using a metabolic pathway that can convert myo-inositol intoglucuronate. In general, such pathways require less enzymatic steps thanthe native metabolic pathways used by plants and animals to produceascorbic acid from glucose. Any method that can efficiently produceascorbic acid from a carbon source such as glucose or phytic acid wouldbe useful for large-scale production efforts. In addition, the methodsand materials provided herein can be used to produce organic compoundswithout the need of chemical steps such as an acid treatment at hightemperature (e.g., a temperature greater than 60° C.).

In general, one aspect of the invention features a method of providing acell with a polypeptide having myo-inositol oxygenase activity. Themethod includes introducing a nucleic acid molecule into the cell, wherethe nucleic acid molecule encodes the polypeptide, and where the cellexpresses the polypeptide. The cell can be a prokaryotic cell (e.g., aPseudomonas, Bacillus, Lactobacillus, Lactococcus, or Corynebacteriumcell). The cell can be a eukaryotic cell (e.g., a yeast, fungi, insect,or mammalian cell). The cell can be a Saccharomyces, Pichia,Aspergillus, Cryptococcus, Schwanniomyces, Schizosaccharomyces,Spodoptera, Cricetulus, or Homo sapiens cell. The nucleic acid moleculecan be integrated into the genome of the cell. The polypeptide cancontain an amino acid sequence at least about 50 percent identical(e.g., at least about 55, 60, 70, 75, 80, or 90 percent identical) tothe sequence set forth in SEQ ID NO:12, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, or 35. The polypeptide can contain anamino acid sequence at least about 70 percent identical to the sequenceset forth in SEQ ID NO:12, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, or 35. The cell can produce L-ascorbic acid. Thecell can have glucuronate reductase activity. The cell can have1,4-lactone hydroxyacylhydrolase activity, D-glucono-1,5-lactonelactonohydrolase activity, and/or uronolactonase activity. The cell canhave gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidaseactivity, and/or gulono-γ-lactone dehydrogenase activity. The cell canhave phosphatase activity and/or phytase activity. The cell can lackL-gulonate 3-dehydrogenase activity. The cell can contain myo-inositoloxygenase activity with a specific activity greater than 40 mgglucuronic acid per gram dry cell weight per hour. The cell can containmyo-inositol oxygenase activity such that an extract from 1×10⁶ cellscontains a specific activity greater than 150 μg glucuronic acid formedper 10 mg total protein per 10 minutes, where each of the 1×10⁶ cells isthe cell or a progeny of the cell. The nucleic acid molecule can containa promoter that is lactose unresponsive. The polypeptide can lack anN-terminal polyhistidine tag. The polypeptide can lack aglutathione-5-transferase sequence.

In another embodiment, the invention features methods of producingglucaric acid as well as cells capable of producing glucaric acid. Thesemethods involve converting myo-inositol to glucuronic acid andconverting glucuronic acid to glucaric acid. The substrates (e.g.,myo-inositol and glucuronic acid) can be converted to their respectiveproducts using polypeptides or chemical conversions. A “chemicalconversion” as used herein refers to the changing of a substrate to aproduct without the aid of a polypeptide having enzymatic activity.Moreover, these methods can be practiced in vivo, in vitro, or by usingcombinations of in vitro and in vivo steps. When polypeptides are usedto convert glucuronic acid to glucaric acid, the polypeptides can haveeither aldehyde dehydrogenase activity, hexose oxidase activity, oraldehyde oxidase activity. When polypeptides are used to convertmyo-inositol to glucuronic acid, the polypeptides can have myo-inositoloxygenase activity.

In another embodiment, the invention features a cell containing anexogenous nucleic acid molecule, where the exogenous nucleic acidmolecule encodes a polypeptide having myo-inositol oxygenase activity,and where the cell expresses the polypeptide. The cell can be aprokaryotic cell (e.g., a Pseudomonas, Bacillus, Lactobacillus,Lactococcus, or Corynebacterium cell). The cell can be a eukaryotic cell(e.g., a yeast, fungi, insect, or mammalian cell). The cell can be aSaccharomyces, Pichia, Aspergillus, Cryptococcus, Schwanniomyces,Schizosaccharomyces, Spodoptera, Cricetulus, or Homo sapiens cell. Thepolypeptide can contain an amino acid sequence at least about 50 percentidentical (e.g., at least about 55, 60, 70, 75, 80, or 90 percentidentical) to the sequence set forth in SEQ ID NO:12, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. The polypeptidecan contain an amino acid sequence at least about 70 percent identicalto the sequence set forth in SEQ ID NO:12, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. The cell can contain a secondexogenous nucleic acid molecule, where the second exogenous nucleic acidmolecule encodes a second polypeptide, and where the cell expresses thesecond polypeptide. The second polypeptide can have glucuronatereductase activity. The second polypeptide can contain an amino acidsequence at least about 50 percent identical to the amino acid sequenceset forth in SEQ ID NO:36. The second polypeptide can have 1,4-lactonehydroxyacylhydrolase activity, D-glucono-1,5-lactone lactonohydrolaseactivity, or uronolactonase activity. The second polypeptide can containan amino acid sequence at least about 50 percent identical to the aminoacid sequence set forth in SEQ ID NO:37 or 38. The second polypeptidecan have gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidaseactivity, or gulono-γ-lactone dehydrogenase activity. The secondpolypeptide can contain an amino acid sequence at least about 50 percentidentical to the amino acid sequence set forth in SEQ ID NO:39 or 40.The second polypeptide can have phosphatase activity. The secondpolypeptide can contain an amino acid sequence at least about 50 percentidentical to the amino acid sequence set forth in SEQ ID NO:41 or 44.The second polypeptide can have phytase activity. The second polypeptidecan contain an amino acid sequence at least about 50 percent identicalto the amino acid sequence set forth in SEQ ID NO:42 or 43. The cell cancontain a second exogenous nucleic acid molecule and a third exogenousnucleic acid molecule, where the second exogenous nucleic acid moleculeencodes a second polypeptide, where the third exogenous nucleic acidmolecule encodes a third polypeptide, and where the cell expresses thesecond polypeptide and the third polypeptide. The second polypeptide canhave glucuronate reductase activity, 1,4-lactone hydroxyacylhydrolaseactivity, D-glucono-1,5-lactone lactonohydrolase activity,gulono-γ-lactone oxidase activity, gulono-γ-lactone dehydrogenaseactivity, uronolactonase activity, galactono-γ-lactone oxidase activity,pyridine nucleotide transhydrogenase activity, phytase, and/orphosphatase activity. The third polypeptide can have glucuronatereductase activity, 1,4-lactone hydroxyacylhydrolase activity,D-glucono-1,5-lactone lactonohydrolase activity, gulono-γ-lactoneoxidase activity, gulono-γ-lactone dehydrogenase activity,uronolactonase activity, galactono-γ-lactone oxidase activity, pyridinenucleotide transhydrogenase activity, phytase activity, and/orphosphatase activity. The cell can lack L-gulonate 3-dehydrogenaseactivity. The cell can have a genetic modification that reducesL-gulonate 3-dehydrogenase activity. The genetic modification caninclude a nucleic acid deletion in the genome of the cell. The cell canproduce ascorbic acid. The cell can have pyridine nucleotidetranshydrogenase activity. The cell can have myo-inositol oxygenaseactivity with a specific activity greater than 40 mg glucuronic acid pergram dry cell weight per hour. The cell can have myo-inositol oxygenaseactivity such that an extract from 1×10⁶ cells comprises a specificactivity greater than 150 μg glucuronic acid formed per 10 mg totalprotein per 10 minutes, where each of the 1×10⁶ cells is the cell or aprogeny of the cell. The exogenous nucleic acid molecule can contain apromoter that is lactose unresponsive. The polypeptide can lack anN-terminal polyhistidine tag. The polypeptide can lack aglutathione-S-transferase sequence. The exogenous nucleic acid moleculecan be integrated into the genome of the cell.

In another aspect, the invention features a method of reducingmyo-inositol oxygenase activity in a cell. The method includesgenetically modifying the genome of the cell such that the expression ofa polypeptide having the myo-inositol oxygenase activity is reduced. Thecell can be a eukaryotic cell (e.g., a plant cell). The geneticmodification can contain a nucleic acid deletion in the genome of thecell.

Another embodiment of the invention features a cell containing a geneticmodification that reduces myo-inositol oxygenase activity. The cell canbe a eukaryotic cell (e.g., a plant cell). The genetic modification caninclude a nucleic acid deletion in the genome of the cell. The cell canlack the myo-inositol oxygenase activity.

Another embodiment of the invention features a cell containing a geneticmodification that reduces L-gulonate 3-dehydrogenase activity. The cellcan be a eukaryotic cell. The genetic modification can include a nucleicacid deletion in the genome of the cell. The cell can lack theL-gulonate 3-dehydrogenase activity.

Another aspect of the invention features an isolated nucleic acidmolecule containing a nucleic acid sequence at least about 50 percentidentical to the sequence set forth in SEQ ID NO: 1. The isolatednucleic acid molecule can encode a polypeptide having myo-inositoloxygenase activity. The nucleic acid sequence can be as set forth in SEQID NO: 1.

In another embodiment, the invention features an isolated nucleic acidmolecule that encodes a polypeptide having an amino acid sequence atleast about 50 percent identical to the sequence set forth in SEQ ID NO:19. The polypeptide can have myo-inositol oxygenase activity. The aminoacid sequence can be as set forth in SEQ ID NO:19.

Another aspect of the invention features a method for producing ascorbicacid. The method includes (a) contacting myo-inositol with a firstpolypeptide having myo-inositol oxygenase activity to form glucuronate,where the first polypeptide is within a cell, (b) contacting theglucuronate with a second polypeptide having glucuronate reductaseactivity to form gulonate, (c) contacting the gulonate with a thirdpolypeptide to form gulono-γ-lactone, the third polypeptide having1,4-lactone hydroxyacylhydrolase activity and/or D-glucono-1,5-lactonelactonohydrolase activity, and (d) contacting the gulono-γ-lactone witha fourth polypeptide to form the ascorbic acid, the fourth polypeptidehaving gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidaseactivity, and/or gulono-γ-lactone dehydrogenase activity, where at least10 mg (e.g., at least 20, 30, 40, 50, 100, or more mg) of ascorbic acidis produced per gram dry cell weight per hour.

Another embodiment of the invention features a method for producingascorbic acid. The method includes (a) contacting myo-inositol with afirst polypeptide having myo-inositol oxygenase activity to formglucuronate, where the first polypeptide is within a cell, (b)contacting the glucuronate with a second polypeptide havinguronolactonase activity to form glucurono-lactone, (c) contacting theglucurono-lactone with a third polypeptide having glucuronolactonereductase activity to form gulono-γ-lactone, and (d) contacting thegulono-γ-lactone with a fourth polypeptide to form the ascorbic acid,the fourth polypeptide having gulono-γ-lactone oxidase activity,galactono-γ-lactone oxidase activity, and/or gulono-γ-lactonedehydrogenase activity, where at least 10 mg (e.g., at least 20, 30, 40,50, 100, or more mg) of ascorbic acid is produced per gram dry cellweight per hour.

Another embodiment of the invention features a method for producingascorbic acid. The method includes (a) contacting myo-inositol with afirst polypeptide having myo-inositol oxygenase activity to formglucuronate, where the first polypeptide is extracellular, (b)contacting the glucuronate with a second polypeptide having glucuronatereductase activity to form gulonate, (c) contacting the gulonate with athird polypeptide to form gulono-γ-lactone, the third polypeptide having1,4-lactone hydroxyacylhydrolase activity and/or D-glucono-1,5-lactonelactonohydrolase activity, and (d) contacting the gulono-γ-lactone witha fourth polypeptide to form the ascorbic acid, the fourth polypeptidehaving gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidaseactivity, and/or gulono-γ-lactone dehydrogenase activity.

Another embodiment of the invention features a method for producingascorbic acid. The method includes (a) contacting myo-inositol with afirst polypeptide having myo-inositol oxygenase activity to formglucuronate, where the first polypeptide is extracellular, (b)contacting the glucuronate with a second polypeptide havinguronolactonase activity to form glucurono-lactone, (c) contacting theglucurono-lactone with a third polypeptide having glucuronolactonereductase activity to form gulono-γ-lactone, and (d) contacting thegulono-γ-lactone with a fourth polypeptide to form the ascorbic acid,the fourth polypeptide having gulono-γ-lactone oxidase activity,galactono-γ-lactone oxidase activity, and/or gulono-γ-lactonedehydrogenase activity.

Glucaric acid, containing two carboxylic acid functional groups, ispotentially useful as an acidulent in the food and animal feedindustries. Glucaric acid has also been shown to be useful as achelating agent and can be used as a biodegradable detergent and anadditive for cement. Glucaric acid, because it is a potent inhibitor ofthe enzyme beta-glucuronidase, has also been shown to be valuable as ananti-cancer agent and has been shown to lower serum cholesterol inmammals. Natural sources with particularly high levels of glucaric acidinclude fruits such as apples and grapefruit and vegetables such asbrussel sprouts and broccoli. Because of its metal chelating properties,it can be used as a chelating agent of 99Tcm for the detection ofmyocardial infarction and the radio-imaging of tumors. It also is a rawmaterial for the production of polyhydroxylated polymers and as such canbe used for the production of fibers, films, and adhesives.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram depicting a seven step metabolic pathway that canproduce ascorbic acid from glucose using D-myo-inositol and L-gulonateas intermediates.

FIG. 2 is a diagram depicting a seven step metabolic pathway that canproduce ascorbic acid from glucose using D-myo-inositol andD-glucurono-3,6-lactone as intermediates.

FIG. 3 is a diagram depicting an eight step metabolic pathway that canproduce ascorbic acid from glucose using UDP-D-glucuronate andL-gulonate as intermediates.

FIG. 4 is a diagram depicting an eight step metabolic pathway that canproduce ascorbic acid from glucose using UDP-D-glucuronate andD-glucurono-3,6-lactone as intermediates.

FIG. 5 is a diagram depicting a nine step metabolic pathway that canproduce ascorbic acid from glucose using UDP-D-glucuronate andL-gulonate as intermediates.

FIG. 6 is a diagram depicting a nine step metabolic pathway that canproduce ascorbic acid from glucose using UDP-D-glucuronate andD-glucurono-3,6-lactone as intermediates.

FIG. 7 is an alignment of 17 amino acid sequences.

FIG. 8 is a graph plotting μg glucuronate formed per assay versus μgprotein per assay for the indicated cell extracts.

FIG. 9 is a sequence listing containing an amino acid sequence from apolypeptide having glucuronate reductase activity. The threonine atposition number 2 can be an alanine.

FIG. 10 is a sequence listing containing an amino acid sequence from apolypeptide having D-glucono-1,5-lactone lactonohydrolase activity. Thethreonine at position number 2 can be an alanine; the valine at position16 can be an alanine; the methionine at position 17 can be anisoleucine; the glutamic acid at position 34 can be a glutamine; and avaline can be inserted after the valine at position 162.

FIG. 11 is a sequence listing containing an amino acid sequence from apolypeptide having uronolactonase activity. The amino acid residues fromposition number 2 through position number 20 can be removed to form amature polypeptide.

FIG. 12 is a sequence listing containing an amino acid sequence from apolypeptide having gulono-γ-lactone oxidase activity. The isoleucine atposition number 85 can be an valine, and the glutamine at position 189can be a histidine.

FIG. 13 is a sequence listing containing an amino acid sequence from apolypeptide having galactono-γ-lactone oxidase activity.

FIG. 14 is a sequence listing containing an amino acid sequence from apolypeptide having acid phosphatase activity.

FIG. 15 is a sequence listing containing an amino acid sequence from apolypeptide having phytase activity.

FIG. 16 is a sequence listing containing an amino acid sequence from apolypeptide having phytase activity.

FIG. 17 is a sequence listing containing an amino acid sequence from apolypeptide having phosphatase activity.

FIG. 18 is a diagram depicting a metabolic pathway that can produceD-glucaric acid from glucose or phytate.

FIG. 19 is a graph plotting μg ascorbic acid per mL per OD600 unit forthe indicated samples after a zero, three, or six hour incubation.

FIG. 20 is a graph plotting the concentration of ascorbic acid for theindicated samples after a zero, four, eight, or nineteen hourincubation.

FIG. 21 contains HPLC and mass spectrometry graphs demonstrating theconversion of glucuronic acid into glucaric acid.

DETAILED DESCRIPTION

The invention provides methods and materials related to producingorganic compounds such as myo-inositol and ascorbic acid. Specifically,the invention provides cells, methods for culturing cells, isolatednucleic acid molecules, and methods and materials for producing variousorganic compounds. In addition, the invention provides several metabolicpathways that can be used to produce ascorbic acid.

1. Seven Step Metabolic Pathways

The invention provides several seven step metabolic pathways that canproduce ascorbic acid from glucose (FIGS. 1 and 2). As depicted in stepone of FIG. 1, D-glucose can be converted into D-glucose-6-phosphate bya polypeptide having either hexokinase activity (EC 2.7.1.1) orglucokinase activity (EC 2.7.1.2). Polypeptides having hexokinaseactivity as well as nucleic acid encoding such polypeptides can beobtained from various species including, without limitation, Homosapiens, Rattus norvegicus, Saccharomyces cerevisiae, Arabidopsisthaliana, and Aspergillus niger. Polypeptides having glucokinaseactivity as well as nucleic acid encoding such polypeptides can beobtained from various species including, without limitation, Bos taurus,Rattus norvegicus, Mus musculus, Homo sapiens, Saccharomyces cerevisiae,Schistosoma mansoni, Aspergillus nidulans, Schizosaccharomyces pombe,Arabidopsis thaliana, Kluyveromyces lactis, Schwanniomyces occidentalis(also known as Debaryomyces occidentalis), Plasmodium falciporum,Bacillus subtilis, Aspergillus niger, Staphylococcus xylosus, Brucellaabortus, Zymomonas mobilis, Escherichia coli, and Streptomycescoelicolor. For example, nucleic acid that encodes a polypeptide havingglucokinase activity can be obtained from Aspergillus niger and can havea sequence as set forth in GenBank® Accession Number X99626.

Alternatively, D-glucose can be converted into D-glucose-6-phosphate bya polypeptide having either polyphosphate:D-glucose 6-phosphotransferaseactivity (EC 2.7.1.63) or D-glucose-6-phosphate phosphohydrolaseactivity (EC 3.1.3.9), or extracellular D-glucose can be transportedinto a cell and converted into D-glucose-6-phosphate by a polypeptidehaving protein-N(pai)-phosphohistidine-sugar phosphotransferase activity(EC 2.7.1.69). Polypeptides having polyphosphate:D-glucose6-phosphotransferase activity as well as nucleic acid encoding suchpolypeptides can be obtained from various species including, withoutlimitation, Mycobacterium tuberculosis. Polypeptides havingD-glucose-6-phosphate phosphohydrolase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, without limitation, Rattus norvegicus and Homo sapiens. Forexample, nucleic acid that encodes a polypeptide havingD-glucose-6-phosphate phosphohydrolase activity can be obtained fromRattus norvegicus and can have a sequence as set forth in GenBank®Accession Number U07993. Polypeptides havingprotein-N(pai)-phosphohistidine-sugar phosphotransferase activity aswell as nucleic acid encoding such polypeptides can be obtained fromvarious species including, without limitation, Escherichia coli andBacillus subtilis.

In step two, the resulting D-glucose-6-phosphate can be converted intoD-myo-inositol-1-phosphate by a polypeptide havingmyo-inositol-1-phosphate synthase activity (EC 5.5.1.4). Polypeptideshaving myo-inositol-1-phosphate synthase activity as well as nucleicacid encoding such polypeptides can be obtained from various speciesincluding, without limitation, Arabidopsis thaliana, Saccharomycescerevisiae, Citrus paradisi, Candida albicans, and Spirodela polyrrhiza.For example, nucleic acid that encodes a polypeptide havingmyo-inositol-1-phosphate synthase activity can be obtained fromSaccharomyces cerevisiae and can have a sequence as set forth inGenBank® Accession Number J04453.

In step three, D-myo-inositol-1-phosphate can be converted intoD-myo-inositol by a polypeptide having myo-inositol-1 (or 4)monophosphatase activity (EC 3.1.3.25). Polypeptides havingmyo-inositol-1 (or 4) monophosphatase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, without limitation, Homo sapiens, Bos taurus, Mus musculus,Rattus norvegicus, Lycopersicon esculentum, Xenopus laevis, andMesembryanthemum crystallinum. For example, nucleic acid that encodes apolypeptide having myo-inositol-1 (or 4) monophosphatase activity can beobtained from Homo sapiens and can have a sequence as set forth inGenBank® Accession Number NM_(—)005536.

In step four, the resulting D-myo-inositol can be converted intoD-glucuronate by a polypeptide having myo-inositol oxygenase activity(EC 1.13.99.1). Polypeptides having myo-inositol oxygenase activity aswell as nucleic acid encoding such polypeptides can be obtained fromvarious species including, without limitation, Rallus norvegicus, Susscrofa, Bos taurus, Cryptococcus neoformans, Schwanniomycesoccidentalis, Homo sapiens, Avena saliva, Pinus radiata, Cryptococcusterreus, Arabidopsis thaliana, and Pleurotus ostreatus.

In step five, D-glucuronate can be converted into L-gulonate by apolypeptide having glucuronate reductase activity (EC 1.1.1.19).Polypeptides having glucuronate reductase activity as well as nucleicacid encoding such polypeptides can be obtained from various speciesincluding, without limitation, Rattus norvegicus, Sus scrofa, and Bostaurus.

In step six, the resulting L-gulonate can be converted intoL-gulono-γ-lactone by a polypeptide having 1,4-lactonehydroxyacylhydrolase activity (EC 3.1.1.25), D-glucono-1,5-lactonelactonohydrolase activity (EC 3.1.1.17), or uronolactonase activity(E.C. 3.1.1.19). Polypeptides having 1,4-lactone hydroxyacylhydrolaseactivity as well as nucleic acid encoding such polypeptides can beobtained from various species including, without limitation, Homosapiens and Ratius norvegicus. Polypeptides having D-glucono-1,5-lactonelactonohydrolase activity as well as nucleic acid encoding suchpolypeptides can be obtained from various species including, withoutlimitation, Zymomonas mobilis, Escherichia coli, Saccharomycescerevisiae, Aspergillus niger, Rattus norvegicus, Sus scrofa, and Bostaurus. For example, nucleic acid that encodes a polypeptide havingD-glucono-1,5-lactone lactonohydrolase activity can be obtained fromZymomonas mobilis and can have a sequence as set forth in GenBank®Accession Number X67189. Polypeptides having uronolactonase activity aswell as nucleic acid encoding such polypeptides can be obtained fromvarious species including, without limitation, Fusarium oxysporum,Aryctolagus cuniculas, Cavia parcellus, Canis familiaris, Macacaphilippinensis, Rattus norvegicus, Sus scrofa, and Bos taurus. Forexample, nucleic acid that encodes a polypeptide having uronolactonaseactivity can be obtained from Fusarium oxysporum and can encode asequence as set forth in GenBank® Accession Number BAA34218.

In step seven, L-gulono-γ-lactone can be converted into L-ascorbate by apolypeptide having gulono-γ-lactone oxidase activity (EC 1.1.3.8), apolypeptide having galactono-γ-lactone oxidase activity (EC 1.1.3.24),or a polypeptide having gulono-γ-lactone dehydrogenase activity.Polypeptides having gulono-γ-lactone oxidase activity as well as nucleicacid encoding such polypeptides can be obtained from various speciesincluding, without limitation, Ratius norvegicus, Tachyglossusaculeatus, Ornithorhynchus anatinus, Perameles nasuta, Isoodonmacrourus, Macropus rufogiseus, Thylogale thetis, Limulus polyphemus,Gallus gallus, Rana catesbeiana, Capra hircus, and Mus musculus. Forexample, nucleic acid that encodes a polypeptide having gulono-γ-lactoneoxidase activity can be obtained from rat and can encode a sequence asset forth in GenBank® Accession Number P10867. Polypeptides havinggalactono-γ-lactone oxidase activity as well as nucleic acid encodingsuch polypeptides can be obtained from various species including,without limitation, Saccharomyces cerevisiae. For example, nucleic acidthat encodes a polypeptide having galactono-γ-lactone oxidase activitycan be obtained from Saccharomyces cerevisiae and can encode a sequenceas set forth in GenBank® Accession Number BAA23804. Polypeptides havinggulono-γ-lactone dehydrogenase activity as well as nucleic acid encodingsuch polypeptides can be obtained from various species including,without limitation, Euglena gracilis (See, e.g., U.S. Pat. No.5,250,428).

The seven step metabolic pathway depicted in FIG. 2 is identical to thepathway depicted in FIG. 1 except that, in step five, D-glucuronate canbe converted into D-glucurono-3,6-lactone by a polypeptide having either1,4-lactone hydroxyacylhydrolase activity (EC 3.1.1.25) orD-glucono-1,5-lactone lactonohydrolase activity (EC 3.1.1.17) oruronolactonase activity (EC 3.1.1.19) and, in step six, the resultingD-glucurono-3,6-lactone can be converted into L-gulono-γ-lactone by apolypeptide having glucuronolactone reductase activity (EC 1.1.1.20).Polypeptides having uronolactonase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, without limitation, Fusarium oxysporum, Oryctolaguscuniculus, Cavia porcellus, Canis familiaris, Macaca philippinensis,Rattus norvegicus, Sus scrofa, and Bos taurus, while polypeptides havingglucuronolactone reductase activity as well as nucleic acid encodingsuch polypeptides can be obtained from various species including,without limitation, Rattus norvegicus. For example, nucleic acid thatencodes a polypeptide having uronolactonase activity can be obtainedfrom Fusarium oxysporum and can encode a sequence as set forth inGenBank® Accession Number BAA34218.

2. Eight Step Metabolic Pathways

The invention provides several eight step metabolic pathways that canproduce ascorbic acid from glucose (FIGS. 3 and 4). As depicted in stepone of FIG. 3, D-glucose can be converted into D-glucose-6-phosphate inthe same manner as described in the seven step metabolic pathwaysdepicted in FIGS. 1 and 2. For example, D-glucose can be converted intoD-glucose-6-phosphate by a polypeptide having hexokinase activity (EC2.7.1.1), glucokinase activity (EC 2.7.1.2), polyphosphate:D-glucose6-phosphotransferase activity (EC 2.7.1.63), or D-glucose-6-phosphatephosphohydrolase activity (EC 3.1.3.9), or extracellular D-glucose canbe transported into a cell and converted into D-glucose-6-phosphate by apolypeptide having protein-N(pai)-phosphohistidine-sugarphosphotransferase activity (EC 2.7.1.69).

In step two, the resulting D-glucose-6-phosphate can be converted intoD-glucose-1-phosphate by a polypeptide having phosphoglucomutaseactivity (EC 5.4.2.2). Polypeptides having phosphoglucomutase activityas well as nucleic acid encoding such polypeptides can be obtained fromvarious species including, without limitation, Arabidopsis thaliana,Homo sapiens, Saccharomyces cerevisiae, and Xanthomonas campestris. Forexample, nucleic acid that encodes a polypeptide havingphosphoglucomutase activity can be obtained from Saccharomycescerevisiae and can have a sequence as set forth in GenBank® AccessionNumber X72016.

In step three, D-glucose-1-phosphate can be converted into UDP-D-glucoseby a polypeptide having UTP-glucose-1-phosphate uridylyltransferaseactivity (EC 2.7.7.9). Polypeptides having UTP-glucose-1-phosphateuridylyltransferase activity as well as nucleic acid encoding suchpolypeptides can be obtained from various species including, withoutlimitation, Bos taurus, Solanuni tuberosum, Pseudomonas aeruginosa,Bacillus subtilis, and Escherichia coli. For example, nucleic acid thatencodes a polypeptide having UTP-glucose-1-phosphate uridylyltransferaseactivity can be obtained from Bos taurus and can have a sequence as setforth in GenBank® Accession Number L14019.

In step four, the resulting UDP-D-glucose can be converted intoUDP-D-glucuronate by a polypeptide having UDP-glucose dehydrogenaseactivity (EC 1.1.1.22). Polypeptides having UDP-glucose dehydrogenaseactivity as well as nucleic acid encoding such polypeptides can beobtained from various species including, without limitation, Homosapiens, Bos taurus, Mus musculus, Drosophila melanogaster, andPseudomonas aeruginosa. For example, nucleic acid that encodes apolypeptide having UDP-glucose dehydrogenase activity can be obtainedfrom Pseudomonas aeruginosa and can have a sequence as set forth inGenBank® Accession Number AJ010734.

In step five, the resulting UDP-D-glucuronate can be converted intoD-glucuronate by a polypeptide having UDP-glucuronateβ-D-glucuronosyltransferase activity (EC 2.4.1.17). Polypeptides havingUDP-glucuronate β-D-glucuronosyltransferase activity as well as nucleicacid encoding such polypeptides can be obtained from various speciesincluding, without limitation, Homo sapiens, Rattus norvegicus, and Musmusculus. For example, nucleic acid that encodes a polypeptide havingUDP-glucuronate β-D-glucuronosyltransferase activity can be obtainedfrom Homo sapiens and can have a sequence as set forth in GenBank®Accession Number NM_(—)001072.

In step six, D-glucuronate can be converted into L-gulonate by apolypeptide having glucuronate reductase activity (EC 1.1.1.19).Polypeptides having glucuronate reductase activity as well as nucleicacid encoding such polypeptides can be obtained from various speciesincluding, without limitation, Rattus norvegicus, Sus scrofa, and Bostaurus.

In step seven, the resulting L-gulonate can be converted intoL-gulono-γ-lactone by a polypeptide having either 1,4-lactonehydroxyacylhydrolase activity (EC 3.1.1.25) or D-glucono-1,5-lactonelactonohydrolase activity (EC 3.1.1.17) or uronolactonase activity (EC3.1.1.19). Polypeptides having 1,4-lactone hydroxyacylhydrolase activityas well as nucleic acid encoding such polypeptides can be obtained fromvarious species including, without limitation, Rattus norvegicus andHomo sapiens, while polypeptides having D-glucono-1,5-lactonelactonohydrolase activity as well as nucleic acid encoding suchpolypeptides can be obtained from various species including, withoutlimitation, Zymomonas mobilis, Escherichia coli, Saccharomycescerevisiae, Aspergillus niger, Rattus norvegicus, Sus scrofa, and Boslaurus. For example, nucleic acid that encodes a polypeptide havingD-glucono-1,5-lactone lactonohydrolase activity can be obtained fromZymomonas mobilis and can have a sequence as set forth in GenBank®Accession Numbers X67189 and S53050.

In step eight, L-gulono-γ-lactone can be converted into L-ascorbate by apolypeptide having gulono-γ-lactone oxidase activity (EC 1.1.3.8), apolypeptide having galactono-γ-lactone oxidase activity (EC 1.1.3.24),or a polypeptide having gulono-γ-lactone dehydrogenase activity.Polypeptides having gulono-γ-lactone oxidase activity as well as nucleicacid encoding such polypeptides can be obtained from various speciesincluding, without limitation, Rattus norvegicus, Tachyglossusaculeatus, Ornithorhynchus anatinus, Perameles nasuta, Isoodonmacrourus, Macropus rufogiseus, Thylogale thetis, Limulus polyphemus,Gallus gallus, Rana catesbeiana, and Capra hircus. For example, nucleicacid that encodes a polypeptide having gulono-γ-lactone oxidase activitycan be obtained from rat and can encode a sequence as set forth inGenBank® Accession Number P10867. Polypeptides havinggalactono-γ-lactone oxidase activity as well as nucleic acid encodingsuch polypeptides can be obtained from various species including,without limitation, Saccharomyces cerevisiae. For example, nucleic acidthat encodes a polypeptide having galactono-γ-lactone oxidase activitycan be obtained from Saccharomyces cerevisiae and can encode a sequenceas set forth in GenBank® Accession Number BAA23804. Polypeptides havinggulono-γ-lactone dehydrogenase as well as nucleic acid encoding suchpolypeptides can be obtained from various species including, withoutlimitation, Euglena gracilis (See, e.g., U.S. Pat. No. 5,250,428).

The eight step metabolic pathway depicted in FIG. 4 is identical to thepathway depicted in FIG. 3 except that, in step six, D-glucuronate canbe converted into D-glucurono-3,6-lactone by a polypeptide having either1,4-lactone hydroxyacylhydrolase activity (EC 3.1.1.25) orD-glucono-1,5-lactone lactonohydrolase activity (EC 3.1.1.17) oruronolactonase activity (EC 3.1.1.19) and, in step seven, the resultingD-glucurono-3,6-lactone can be converted into L-gulono-γ-lactone by apolypeptide having glucuronolactone reductase activity (EC 1.1.1.20).

Polypeptides having uronolactonase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, without limitation, Fusarium oxysporum, Oryctolaguscuniculus, Cavia porcellus, Canis familiaris, Macaca philippinensis,Rattus norvegicus, Sus scrofa, and Bos taurus, while polypeptides havingglucuronolactone reductase activity as well as nucleic acid encodingsuch polypeptides can be obtained from various species including,without limitation, Rattus norvegicus.

3. Nine Step Metabolic Pathways

The invention provides several nine step metabolic pathways that canproduce ascorbic acid from glucose (FIGS. 5 and 6). The nine stepmetabolic pathways depicted in FIGS. 5 and 6 are similar to the eightstep metabolic pathways depicted in FIGS. 3 and 4, respectively, exceptthat the conversion of UDP-D-glucuronate into D-glucuronate uses aD-glucuronate-1-phosphate intermediate. As depicted in FIGS. 5 and 6,step five involves the conversion of UDP-D-glucuronate intoD-glucuronate-1-phosphate by a polypeptide having UTP:1-phospho-α-D-glucuronate uridylyltransferase activity (EC 2.7.7.44).Polypeptides having UTP: 1-phospho-α-D-glucuronate uridylyltransferaseactivity as well as nucleic acid encoding such polypeptides can beobtained from various species including, without limitation, Hordeumvulgare and Typha latifolia.

In step six, the resulting D-glucuronate-1-phosphate can be convertedinto D-glucuronate by a polypeptide having ATP:D-glucuronate1-phosphotransferase activity (EC 2.7.1.43). Polypeptides havingATP:D-glucuronate 1-phosphotransferase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, without limitation, Vigna radiata, Nicotiana tabacum, Liliumlongiflorum, Zea mays, and Glycine max.

4. Phytic Acid

The invention provides several pathways that can be used to producemyo-inositol or ascorbic acid from phytic acid. For example, phytic acidcan be converted into myo-inositol by a polypeptide having phytaseactivity, by a polypeptide having phosphatase activity (or a collectionof polypeptides having different phosphatase activities), or a mixtureof polypeptides having phytase activity and polypeptides havingphosphatase activity (or a collection of polypeptides having differentphosphatase activities). For example, a polypeptide having phytaseactivity can be used to convert phytic acid into myo-inositol.Polypeptides having phytase activity as well as nucleic acid encodingsuch polypeptides can be obtained from various species including,without limitation, Schwanniomyces occidentalis, Bacillus subtilis, E.coli, Aspergillus terreus, Homo sapiens, and Zea mays. For example,nucleic acid that encodes a polypeptide having phytase activity can beobtained from E. coli and can have a sequence as set forth in GenBank®Accession Number M58708, or can be obtained from Bacillus subtilis andcan have a sequence as set forth in GenBank® Accession Number AF298179or AI277890. Also, polypeptides having phytase activity as well asnucleic acid encoding such polypeptides can be obtained as described inU.S. Pat. Nos. 5,830,733; 5,840,561; or 5,830,732. In one embodiment, apolypeptide having the sequence set forth in FIG. 16 can be used toconvert phytic acid into myo-inositol. Alternatively, a polypeptidemixture having multiple inositol polyphosphate phosphatase activitiescan be used to convert phytic acid into myo-inositol. Polypeptideshaving phosphatase activity (e.g., acid phosphatase activity) as well asnucleic acid encoding such polypeptides can be obtained from variousspecies including, without limitation, E. coli, Saccharomycescerevisiae, Schizosaccharomyces pombe, Candida albicans, and Aspergillusniger. For example, nucleic acid that encodes a polypeptide havingphosphatase activity can be obtained from E. coli and can encode asequence as set forth in GenBank® Accession Number P07102. Also,polypeptides having phosphatase activity as well as nucleic acidencoding such polypeptides can be obtained as described in U.S. Pat. No.5,830,733. A mixture of polypeptides having phytase activity andpolypeptides having phosphatase activities can be used to convert phyticacid into inositol as described in U.S. Pat. No. 5,830,733. In addition,phytic acid can be converted into myo-inositol using any chemicaltechnique such as heat or steam treatments.

The resulting myo-inositol can be converted into any other organiccompound (e.g., ascorbic acid) using any of the enzymatic stepsdescribed herein. For example, myo-inositol can be converted intoascorbic acid using steps four through seven of the seven step metabolicpathway of FIG. 1.

5. Glucaric Acid

The invention provides pathways that can be used to produce glucaricacid (FIG. 18). For example, glucose or phytic acid can be convertedinto glucuronic acid as described herein. The resulting glucuronic canbe converted into glucaric acid by a polypeptide having non-specifichexose oxidase activity (EC 1.1.3.5), by a polypeptide having aldehydedehydrogenase [NAD(P)] activity (EC 1.2.1.5, EC 1.2.1.3 (NAD), or EC1.2.1.4 (NADP)), or by a polypeptide having aldehyde oxidase activity(EC 1.2.3.1). For example, a polypeptide having non-specific hexoseoxidase activity can be used to convert glucuronic into glucaric acid.Polypeptides having non-specific hexose oxidase activity as well asnucleic acid encoding such polypeptides can be obtained from variousspecies including, without limitation, Chondrus crispus, Yersiniapestis, Yersinia pseudotuberculosis, and Ralstonia solanacearum. Forexample, nucleic acid that encodes a polypeptide having non-specifichexose oxidase activity can be obtained from Chondrus crispus and canencode an amino acid sequence as set forth in GenBank® Accession NumberAAB49376.1, or can be obtained from Yersinia pestis and can encode anamino acid sequence as set forth in GenBank® Accession NumberNP_(—)403959.1, or can be obtained from Ralstonia solanacearum and canencode an amino acid sequence as set forth in GenBank® Accession NumberNP_(—)518171.1. Also, polypeptides having hexose oxidase activity aswell as nucleic acid encoding such polypeptides can be obtained asdescribed elsewhere (U.S. Pat. No. 6,251,626 and Sullivan and Ikawa,Biochimica et Biophysica Acta, 309:11-22 (1973)). In addition,glucuronic can be converted into glucaric acid using any chemicaltechnique such as oxidation using molecular oxygen and a catalyst. Forexample, methods similar to those described in U.S. Pat. No. 5,817,870or Gallezot et al. (Chem. Ind. (Dekker), 62:331-40 (1995)) can be usedto convert glucuronic into glucaric acid.

Additionally, a polypeptide having aldehyde dehydrogenase activity canbe used to convert glucuronic into glucaric acid. Polypeptides havingaldehyde dehydrogenase activity as well as nucleic acid encoding suchpolypeptides can be obtained from various species including, withoutlimitation, Bacillus slearothermophilus (gi:1169292) and Bacillussubtilus (gi:16077316 or NP_(—)388129.1). Similarly, a polypeptidehaving aldehyde oxidase activity can be used to convert glucuronic intoglucaric acid. Polypeptides having aldehyde oxidase activity as well asnucleic acid encoding such polypeptides can be obtained from variousspecies including, without limitation, Oryza sativa (gi: 1844950 orAAL700116.1), Zea mays (BAA23226.1), and Lycopersicon esculentum(AAG22607.1 or AF258810).

6. Nucleic Acid

The invention provides isolated nucleic acid molecules that contain anucleic acid sequence at least about 50 percent identical (e.g., atleast about 55, 65, 70, 75, 80, 85, 90, 95, or 99 percent identical) tothe sequence set forth in SEQ ID NO: 1. The invention also providesisolated nucleic acid molecules that encode a polypeptide having anamino acid sequence at least about 50 percent identical (e.g., at leastabout 55, 65, 70, 75, 80, 85, 90, 95, or 99 percent identical) to thesequence set forth in SEQ ID NO:19.

The term “nucleic acid” as used herein encompasses both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

The term “isolated” as used herein with reference to nucleic acid refersto a naturally-occurring nucleic acid that is not immediately contiguouswith both of the sequences with which it is immediately contiguous (oneon the 5′ end and one on the 3′ end) in the naturally-occurring genomeof the organism from which it is derived. For example, an isolatednucleic acid can be, without limitation, a recombinant DNA molecule ofany length, provided one of the nucleic acid sequences normally foundimmediately flanking that recombinant DNA molecule in anaturally-occurring genome is removed or absent. Thus, an isolatednucleic acid includes, without limitation, a recombinant DNA that existsas a separate molecule (e.g., a cDNA or a genomic DNA fragment producedby PCR or restriction endonuclease treatment) independent of othersequences as well as recombinant DNA that is incorporated into a vector,an autonomously replicating plasmid, a virus (e.g., a retrovirus,adenovirus, or herpes virus), or into the genomic DNA of a prokaryote oreukaryote. In addition, an isolated nucleic acid can include arecombinant DNA molecule that is part of a hybrid or fusion nucleic acidsequence.

The term “isolated” as used herein with reference to nucleic acid alsoincludes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in a naturallyoccurring genome. For example, non-naturally-occurring nucleic acid suchas an engineered nucleic acid is considered to be isolated nucleic acid.Engineered nucleic acid can be made using common molecular cloning orchemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence.

It will be apparent to those of skill in the art that a nucleic acidexisting among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest is not to be considered an isolatednucleic acid.

The term “exogenous” as used herein with reference to nucleic acid and aparticular cell refers to any nucleic acid that does not originate fromthat particular cell as found in nature. Thus, allnon-naturally-occurring nucleic acid is considered to be exogenous to acell once introduced into the cell. It is important to note thatnon-naturally-occurring nucleic acid can contain nucleic acid sequencesor fragments of nucleic acid sequences that are found in nature providedthe nucleic acid as a whole does not exist in nature. For example, anucleic acid molecule containing a genomic DNA sequence within anexpression vector is non-naturally-occurring nucleic acid, and thus isexogenous to a cell once introduced into the cell, since that nucleicacid molecule as a whole (genomic DNA plus vector DNA) does not exist innature. Thus, any vector, autonomously replicating plasmid, or virus(e.g., retrovirus, adenovirus, or herpes virus) that as a whole does notexist in nature is considered to be non-naturally-occurring nucleicacid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid.

Nucleic acid that is naturally occurring can be exogenous to aparticular cell. For example, an entire chromosome isolated from a cellof person X is an exogenous nucleic acid with respect to a cell ofperson Y once that chromosome is introduced into Y's cell.

The percent identity between a particular nucleic acid or amino acidsequence and a sequence referenced by a particular sequenceidentification number is determined as follows. First, a nucleic acid oramino acid sequence is compared to the sequence set forth in aparticular sequence identification number using the BLAST 2 Sequences(B12seq) program from the stand-alone version of BLASTZ containingBLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-aloneversion of BLASTZ can be obtained from Fish & Richardson's web site(e.g., www.fr.com/blast) or the United States government's NationalCenter for Bio/technology Information web site (e.g.,www.ncbi.nlm.nih.gov). Instructions explaining how to use the B12seqprogram can be found in the readme file accompanying BLASTZ. B12seqperforms a comparison between two sequences using either the BLASTN orBLASTP algorithm. BLASTN is used to compare nucleic acid sequences,while BLASTP is used to compare amino acid sequences. To compare twonucleic acid sequences, the options are set as follows: −i is set to afile containing the first nucleic acid sequence to be compared (e.g.,C:\seq1.txt); −j is set to a file containing the second nucleic acidsequence to be compared (e.g., C:\seq2.txt); −p is set to blastn; −o isset to any desired file name (e.g., C:\output.txt); −q is set to −1; −ris set to 2; and all other options are left at their default setting.For example, the following command can be used to generate an outputfile containing a comparison between two sequences: C:\B12seq −ic:\seq1.txt −j c:\seq2.txt −p blastn −o c:\output.txt −q −1 −r 2. Tocompare two amino acid sequences, the options of B12seq are set asfollows: −i is set to a file containing the first amino acid sequence tobe compared (e.g., C:\seq1.txt); −j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); −p is setto blastp; −o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\B12seq −i c:\seq1.txt −jc:\seq2.txt −p blastp −o c:\output.txt. If the two compared sequencesshare homology, then the designated output file will present thoseregions of homology as aligned sequences. If the two compared sequencesdo not share homology, then the designated output file will not presentaligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent identity is determined bydividing the number of matches by the length of the sequence set forthin the identified sequence (e.g., SEQ ID NO: 1) followed by multiplyingthe resulting value by 100. For example, a nucleic acid sequence thathas 711 matches when aligned with the sequence set forth in SEQ ID NO: 1is 75 percent identical to the sequence set forth in SEQ ID NO: 1 (i.e.,711÷948*100=75).

It is noted that the percent identity value is rounded to the nearesttenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2.It also is noted that the length value will always be an integer.

The invention also provides isolated nucleic acid molecules that (1)encode a polypeptide having myo-inositol oxygenase activity and (2)hybridize, under hybridization conditions, to the sense or antisensestrand of a nucleic acid having the sequence set forth in SEQ ID NO: 1.The hybridization conditions can be moderately or highly stringenthybridization conditions.

For the purpose of this invention, moderately stringent hybridizationconditions mean the hybridization is performed at about 42° C. in ahybridization solution containing 25 mM KPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50%formamide, 10% dextran sulfate, and 1-15 ng/mL probe (about 5×10⁷cpm/μg), while the washes are performed at about 50° C. with a washsolution containing 2×SSC and 0.1% sodium dodecyl sulfate.

Highly stringent hybridization conditions mean the hybridization isperformed at about 42° C. in a hybridization solution containing 25 mMKPO₄ (pH 7.4), 5×SSC, 5× Denhart's solution, 50 μg/mL denatured,sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15ng/mL probe (about 5×10⁷ cpm/μg), while the washes are performed atabout 65° C. with a wash solution containing 0.2×SSC and 0.1% sodiumdodecyl sulfate.

Isolated nucleic acid molecules within the scope of the invention can beobtained using any method including, without limitation, commonmolecular cloning and chemical nucleic acid synthesis techniques. Forexample, PCR can be used to obtain an isolated nucleic acid moleculecontaining a nucleic acid sequence sharing similarity to the sequenceset forth in SEQ ID NO:1. PCR refers to a procedure or technique inwhich target nucleic acid is amplified in a manner similar to thatdescribed in U.S. Pat. No. 4,683,195, and subsequent modifications ofthe procedure described therein. Generally, sequence information fromthe ends of the region of interest or beyond are used to designoligonucleotide primers that are identical or similar in sequence toopposite strands of a potential template to be amplified. Using PCR, anucleic acid sequence can be amplified from RNA or DNA. For example, anucleic acid sequence can be isolated by PCR amplification from totalcellular RNA, total genomic DNA, and cDNA as well as from bacteriophagesequences, plasmid sequences, viral sequences, and the like. When usingRNA as a source of template, reverse transcriptase can be used tosynthesize complimentary DNA strands.

Isolated nucleic acid molecules within the scope of the invention alsocan be obtained by mutagenesis. For example, an isolated nucleic acidcontaining a sequence set forth in SEQ ID NO: 1 can be mutated usingcommon molecular cloning techniques (e.g., site-directed mutagenesis).Possible mutations include, without limitation, deletions, insertions,and substitutions, as well as combinations of deletions, insertions, andsubstitutions.

In addition, nucleic acid and amino acid databases (e.g., GenBank®) canbe used to obtain an isolated nucleic acid molecule within the scope ofthe invention. For example, any nucleic acid sequence having somehomology to a sequence set forth in SEQ ID NO: 1, or any amino acidsequence having some homology to a sequence set forth in SEQ ID NO: 19can be used as a query to search GenBank®.

Further, nucleic acid hybridization techniques can be used to obtain anisolated nucleic acid molecule within the scope of the invention.Briefly, any nucleic acid molecule having some homology to a sequenceset forth in SEQ ID NO: 1 can be used as a probe to identify a similarnucleic acid by hybridization under conditions of moderate to highstringency. Once identified, the nucleic acid molecule then can bepurified, sequenced, and analyzed to determine whether it is within thescope of the invention as described herein.

Hybridization can be done by Southern or Northern analysis to identify aDNA or RNA sequence, respectively, which hybridizes to a probe. Theprobe can be labeled with a biotin, digoxygenin, an enzyme, or aradioisotope such as ³²P. The DNA or RNA to be analyzed can beelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,(1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory,Plainview, N.Y. Typically, a probe is at least about 20 nucleotides inlength. For example, a probe corresponding to a 20-nucleotide sequenceset forth in SEQ ID NO: 1 can be used to identify an identical orsimilar nucleic acid. In addition, probes longer or shorter than 20nucleotides can be used.

7. Genetically Modified Cells

The invention provides genetically modified cells (e.g., cellscontaining an exogenous nucleic acid molecule). Such cells can be usedto produce an organic compound such as ascorbic acid, glucuronic acid,and glucaric acid. The cells can be eukaryotic or prokaryotic. Forexample, genetically modified cells of the invention can be mammaliancells (e.g., human, murine, and bovine cells), plant cells (e.g., corn,wheat, rice, and soybean cells), fungal cells (e.g., yeast cells), orbacterial cells (e.g., E. coli cells). A cell of the invention also canbe a microorganism. The term “microorganism” as used herein refers toall microscopic organisms including, without limitation, bacteria,algae, fungi, and protozoa. Thus, E. coli, S. cerevisiae, Kluyveromyceslactis, A. niger, Cr. terreus, Sch. occidentalis, and Sz. pombe areconsidered microorganisms.

Typically, a cell of the invention is genetically modified such that aparticular organic compound is produced. Such cells can contain one ormore exogenous nucleic acid molecules that encode polypeptides havingenzymatic activity. For example, a microorganism can contain exogenousnucleic acid that encodes a polypeptide having myo-inositol oxygenaseactivity. In this case, D-myo-inositol can be converted intoD-glucuronate which can lead to the production of ascorbic acid. It isnoted that a cell can be given an exogenous nucleic acid molecule thatencodes a polypeptide having an enzymatic activity that catalyzes theproduction of a compound not normally produced by that cell.Alternatively, a cell can be given an exogenous nucleic acid moleculethat encodes a polypeptide having an enzymatic activity that catalyzesthe production of a compound that is normally produced by that cell. Inthis case, the genetically modified cell can produce more of thecompound, or can produce the compound more efficiently, than a similarcell not having the genetic modification.

A polypeptide having a particular enzymatic activity can be apolypeptide that is either naturally occurring or non-naturallyoccurring. A naturally occurring polypeptide is any polypeptide havingan amino acid sequence as found in nature, including wild-type andpolymorphic polypeptides. Such naturally occurring polypeptides can beobtained from any species including, without limitation, mammalian,fungal, and bacterial species. A non-naturally occurring polypeptide isany polypeptide having an amino acid sequence that is not found innature. Thus, a non-naturally occurring polypeptide can be a mutatedversion of a naturally occurring polypeptide or an engineeredpolypeptide. For example, a non-naturally occurring polypeptide havingmyo-inositol oxygenase activity can be a mutated version of a naturallyoccurring polypeptide having myo-inositol oxygenase activity thatretains at least some myo-inositol oxygenase activity. A polypeptide canbe mutated by, for example, sequence additions, deletions, and/orsubstitutions using standard methods such as site-directed mutagenesisof the corresponding nucleic acid coding sequence.

The invention provides genetically modified cells that can be used toperform one or more steps of a metabolic pathway described herein. Forexample, an individual microorganism can contain an exogenous nucleicacid molecule such that each of the polypeptides necessary to performall seven steps of a seven step metabolic pathway is expressed. It isimportant to note that such cells can contain any number of exogenousnucleic acid molecules. For example, a particular cell can contain sevenexogenous nucleic acid molecules with each one encoding one of the sevenpolypeptides necessary to perform a seven step metabolic pathway, or aparticular cell can endogenously produce polypeptides necessary toperform the first six of the seven steps of a seven step metabolicpathway while containing an exogenous nucleic acid molecule that encodesa polypeptide necessary to perform the seventh step. It is noted that acell containing an exogenous nucleic acid molecule that encodes apolypeptide having a particular activity can also endogenously express apolypeptide having a similar activity. In such cases, providing a cellwith an exogenous nucleic acid molecule that encodes a polypeptidehaving an activity similar to an endogenously expressed polypeptide isexpected to provide that cell with enhanced activity as compared to asimilar cell lacking the exogenous nucleic acid molecule. It also isnoted that a cell can contain an exogenous nucleic acid molecule thatencodes a polypeptide having pyridine nucleotide transhydrogenaseactivity. Such a polypeptide can be used to generate NADPH within a cellby catalyzing a chemical reaction (e.g., NADH+NADP→NAD+NADPH). Anysource can be used to obtain a polypeptide having pyridine nucleotidetranshydrogenase activity or a nucleic acid encoding such a polypeptide.For example, nucleic acid encoding a polypeptide having pyridinenucleotide transhydrogenase activity can be obtained as describedelsewhere (e.g., U.S. Pat. No. 5,830,716 and Nissen et al., Yeast18:19-32 (2001)). In addition, a single exogenous nucleic acid moleculecan encode one or more than one polypeptide. For example, a singleexogenous nucleic acid molecule can contain sequences that encode threedifferent polypeptides. Further, the cells described herein can containa single copy, or multiple copies (e.g., about 5, 10, 20, 35, 50, 75,100 or 150 copies), of a particular exogenous nucleic acid molecule. Forexample, a particular cell can contain about 50 copies of an exogenousnucleic acid molecule X. Again, the cells described herein can containmore than one particular exogenous nucleic acid molecule. For example, aparticular cell can contain about 50 copies of exogenous nucleic acidmolecule X as well as about 75 copies of exogenous nucleic acid moleculeY.

In one embodiment, the invention provides a cell containing an exogenousnucleic acid molecule that encodes a polypeptide having enzymaticactivity that leads to the formation of ascorbic acid. It is noted thatthe produced ascorbic acid can be secreted from the cell, eliminatingthe need to disrupt cell membranes to retrieve the organic compound.Typically, the cell of the invention produces the organic compound withthe concentration being at least about 0.1 grams per L (e.g., at leastabout 1 g/L, 5 g/L, 10 g/L, or 80 g/L). When determining the yield oforganic compound production for a particular cell, any method can beused. See, e.g., Kiers et al., Yeast, 14(5):459-469 (1998). Typically, acell within the scope of the invention such as a microorganismcatabolizes a hexose carbon source such as glucose. A cell, however, cancatabolize a variety of carbon sources such as pentose sugars (e.g.,ribose, arabinose, xylose, and lyxose), glycerols, or myo-inositol. Inother words, a cell within the scope of the invention can utilize avariety of carbon sources.

In another embodiment, a cell within the scope of the invention cancontain an exogenous nucleic acid molecule that encodes a polypeptidehaving myo-inositol oxygenase activity. Such cells can have any level ofmyo-inositol oxygenase activity. For example, a cell containing anexogenous nucleic acid molecule that encodes a polypeptide havingmyo-inositol oxygenase activity can have myo-inositol oxygenase activitywith a specific activity greater than about 5 mg glucuronic acid formedper gram dry cell weight per hour (e.g., greater than about 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 500, ormore mg glucuronic acid formed per gram dry cell weight per hour).Alternatively, a cell can have myo-inositol oxygenase activity such thata cell extract from 1×10⁶ cells has a specific activity greater thanabout 5 μg glucuronic acid formed per 10 mg total protein per 10 minutes(e.g., greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125,150, 200, 250, 300, 350, 400, 500, or more μg glucuronic acid formed per10 mg total protein per 10 minutes).

A nucleic acid molecule encoding a polypeptide having enzymatic activitycan be identified and obtained using any method. For example, standardnucleic acid sequencing techniques and software programs that translatenucleic acid sequences into amino acid sequences based on the geneticcode can be used to determine whether or not a particular nucleic acidhas any sequence homology with known enzymatic polypeptides. Sequencealignment software such as MEGALIGN® (DNASTAR, Madison, Wis., 1997) canbe used to compare various sequences. In addition, nucleic acidmolecules encoding known enzymatic polypeptides can be mutated usingcommon molecular cloning techniques (e.g., site-directed mutageneses).Possible mutations include, without limitation, deletions, insertions,and base substitutions, as well as combinations of deletions,insertions, and base substitutions. Further, nucleic acid and amino aciddatabases (e.g., GenBank®) can be used to identify a nucleic acidsequence that encodes a polypeptide having enzymatic activity. Briefly,any amino acid sequence having some homology to a polypeptide havingenzymatic activity, or any nucleic acid sequence having some homology toa sequence encoding a polypeptide having enzymatic activity can be usedas a query to search GenBank®. The identified polypeptides then can beanalyzed to determine whether or not they exhibit enzymatic activity.

Nucleic acid molecules that encode a polypeptide having enzymaticactivity can be identified and obtained using common molecular cloningor chemical nucleic acid synthesis procedures and techniques, includingPCR. PCR refers to a procedure or technique in which target nucleic acidis amplified in a manner similar to that described in U.S. Pat. No.4,683,195, and subsequent modifications of the procedure describedtherein. Generally, sequence information from the ends of the region ofinterest or beyond are used to design oligonucleotide primers that areidentical or similar in sequence to opposite strands of a potentialtemplate to be amplified. Using PCR, a nucleic acid sequence can beamplified from RNA or DNA. For example, a nucleic acid sequence can beisolated by PCR amplification from total cellular RNA, total genomicDNA, and cDNA as well as from bacteriophage sequences, plasmidsequences, viral sequences, and the like. When using RNA as a source oftemplate, reverse transcriptase can be used to synthesize complimentaryDNA strands.

In addition, nucleic acid hybridization techniques can be used toidentify and obtain a nucleic acid molecule that encodes a polypeptidehaving enzymatic activity. Briefly, any nucleic acid molecule thatencodes a known enzymatic polypeptide, or fragment thereof, can be usedas a probe to identify a similar nucleic acid molecules by hybridizationunder conditions of moderate to high stringency. Such similar nucleicacid molecules then can be isolated, sequenced, and analyzed todetermine whether the encoded polypeptide has enzymatic activity.

Hybridization can be done by Southern or Northern analysis to identify aDNA or RNA sequence, respectively, that hybridizes to a probe. The probecan be labeled with a radioisotope such as ³²P, an enzyme, digoxygenin,or by biotinylation. The DNA or RNA to be analyzed can beelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,(1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory,Plainview, N.Y. Typically, a probe is at least about 20 nucleotides inlength. For example, a probe corresponding to a 20 nucleotide sequencethat encodes a mammalian myo-inositol oxygenase can be used to identifya nucleic acid molecule that encodes a fungal polypeptide havingmyo-inositol oxygenase activity. In addition, probes longer or shorterthan 20 nucleotides can be used.

Expression cloning techniques also can be used to identify and obtain anucleic acid molecule that encodes a polypeptide having enzymaticactivity. For example, a substrate known to interact with a particularenzymatic polypeptide can be used to screen a phage display librarycontaining that enzymatic polypeptide. Phage display libraries can begenerated as described elsewhere (Burritt et al., Anal. Biochem. 238:1-13 (1990)), or can be obtained from commercial suppliers such asNovagen (Madison, Wis.).

Further, polypeptide sequencing techniques can be used to identify andobtain a nucleic acid molecule that encodes a polypeptide havingenzymatic activity. For example, a purified polypeptide can be separatedby gel electrophoresis, and its amino acid sequence determined by, forexample, amino acid microsequencing techniques. Once determined, theamino acid sequence can be used to design degenerate oligonucleotideprimers. Degenerate oligonucleotide primers can be used to obtain thenucleic acid encoding the polypeptide by PCR. Once obtained, the nucleicacid can be sequenced, cloned into an appropriate expression vector, andintroduced into a microorganism.

Any method can be used to introduce an exogenous nucleic acid moleculeinto a cell. In fact, many methods for introducing nucleic acid intomicroorganisms such as bacteria and yeast are well known to thoseskilled in the art. For example, heat shock, lipofection,electroporation, conjugation, fusion of protoplasts, and biolisticdelivery are common methods for introducing nucleic acid into bacteriaand yeast cells. See, e.g., Ito et al., J. Bacterol. 153:163-168 (1983);Durrens et al., Curr. Genet. 18:7-12 (1990); and Becker and Guarente,Methods in Enzymology 194:182-187 (1991).

An exogenous nucleic acid molecule contained within a particular cell ofthe invention can be maintained within that cell in any form. Forexample, exogenous nucleic acid molecules can be integrated into thegenome of the cell or maintained in an episomal state. In other words, acell of the invention can be a stable or transient transformant. Inaddition, a microorganism described herein can contain a single copy, ormultiple copies (e.g., about 5, 10, 20, 35, 50, 75, 100 or 150 copies),of a particular exogenous nucleic acid molecule as described above.

Methods for expressing an amino acid sequence from an exogenous nucleicacid molecule are well known to those skilled in the art. Such methodsinclude, without limitation, constructing a nucleic acid such that aregulatory element promotes the expression of a nucleic acid sequencethat encodes a polypeptide. Typically, regulatory elements are DNAsequences that regulate the expression of other DNA sequences at thelevel of transcription. Thus, regulatory elements include, withoutlimitation, promoters, enhancers, and the like. Any type of promoter canbe used to express an amino acid sequence from an exogenous nucleic acidmolecule. Examples of promoters include, without limitation,constitutive promoters, tissue-specific promoters, and promotersresponsive or unresponsive to a particular stimulus (e.g., light,oxygen, chemical concentration, and the like). For example, a promoterthat is unresponsive to lactose can be used to express a polypeptidehaving myo-inositol oxygenase activity. Moreover, methods for expressinga polypeptide from an exogenous nucleic acid molecule in cells such asbacterial cells and yeast cells are well known to those skilled in theart. For example, nucleic acid constructs that are capable of expressingexogenous polypeptides within E. coli are well known. See, e.g.,Sambrook et al., Molecular cloning: a laboratory manual, Cold SpringHarbour Laboratory Press, New York, USA, second edition (1989).

As described herein, a cell within the scope of the invention cancontain an exogenous nucleic acid molecule that encodes a polypeptidehaving enzymatic activity that leads to the formation of ascorbic acid.Methods of identifying cells that contain exogenous nucleic acid arewell known to those skilled in the art. Such methods include, withoutlimitation, PCR and nucleic acid hybridization techniques such asNorthern and Southern analysis. In some cases, immunohistochemistry andbiochemical techniques can be used to determine if a cell contains aparticular nucleic acid by detecting the expression of the encodedenzymatic polypeptide encoded by that particular nucleic acid molecule.For example, an antibody having specificity for an encoded enzyme can beused to determine whether or not a particular cell contains that encodedenzyme. Further, biochemical techniques can be used to determine if acell contains a particular nucleic acid molecule encoding an enzymaticpolypeptide by detecting an organic product produced as a result of theexpression of the enzymatic polypeptide. For example, detection ofascorbic acid after introduction of exogenous nucleic acid that encodesa polypeptide having L-gulono-γ-lactone oxidase activity into a cellthat does not normally express such a polypeptide can indicate that thatcell not only contains the introduced exogenous nucleic acid moleculebut also expresses the encoded enzymatic polypeptide from thatintroduced exogenous nucleic acid molecule. Methods for detectingspecific enzymatic activities or the presence of particular organicproducts are well known to those skilled in the art. For example, thepresence of ascorbic acid can be determined as described elsewhere. See,Sullivan and Clarke, J. Assoc. Offic. Agr. Chemists, 38:514-518 (1955).

The invention also provides genetically modified cells having reducedpolypeptide activity. The term “reduced” as used herein with respect toa cell and a particular polypeptide's activity refers to a lower levelof activity than that measured in a comparable cell of the same species.For example, a particular microorganism lacking enzymatic activity X isconsidered to have reduced enzymatic activity X if a comparablemicroorganism has at least some enzymatic activity X. It is noted that acell can have the activity of any type of polypeptide reduced including,without limitation, enzymes, transcription factors, transporters,receptors, signal molecules, and the like. For example, a cell cancontain an exogenous nucleic acid molecule that disrupts a regulatoryand/or coding sequence of a polypeptide having myo-inositol oxygenaseactivity. Disrupting myo-inositol oxygenase expression can lead to theaccumulation of D-myo-inositol or derivatives. It is also noted thatreduced polypeptide activities can be the result of lower polypeptideconcentration, lower specific activity of a polypeptide, or combinationsthereof. Many different methods can be used to make a cell havingreduced polypeptide activity. For example, a cell can be engineered tohave a disrupted regulatory sequence or polypeptide-encoding sequenceusing common mutagenesis or knock-out technology. See, e.g., Methods inYeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Sterns,Cold Spring Harbor Press (1998). Alternatively, antisense technology canbe used to reduce the activity of a particular polypeptide. For example,a cell can be engineered to contain a cDNA that encodes an antisensemolecule that prevents a polypeptide from being translated. The term“antisense molecule” as used herein encompasses any nucleic acidmolecule or nucleic acid analog (e.g., peptide nucleic acids) thatcontains a sequence that corresponds to the coding strand of anendogenous polypeptide. An antisense molecule also can have flankingsequences (e.g., regulatory sequences). Thus, antisense molecules can beribozymes or antisense oligonucleotides. A ribozyme can have any generalstructure including, without limitation, hairpin, hammerhead, or axheadstructures, provided the molecule cleaves RNA.

A cell having reduced activity of a polypeptide can be identified usingany method. For example, biological assays such as the assay describedin Example 3 for measuring myo-inositol oxygenase activity can be usedto identify cells having reduced myo-inositol oxygenase activity.

In one embodiment, the invention provides microorganisms that containreduced myo-inositol transporter activity. Microorganisms containingreduced myo-inositol transporter activity can produce inositol andinositol-related products (e.g., myo-inositol, meso-inositol,hexahydroxycyclohexane, and Vitamin B₈) in the presence of inositol. Inother words, inositol-1-phosphate synthase activity (EC 5.5.1.4) is notregulated by inositol in microorganisms lacking myo-inositol transporteractivity. In general, such microorganisms can be produced by reducingthe activity of itr1, itr2, opi1, or similar polypeptides. Again,microorganisms containing reduced myo-inositol transporter activity canbe produced by any method including, without limitation, mutagenesis,knock-out, and anti-sense technology. It is noted that nucleic acid thatencodes itr1 from S. cerevisiae is set forth in GenBank® AccessionNumber D90352, and nucleic acid that encodes itr2 from S. cerevisiae isset forth in GenBank® Accession Number D90353.

In another embodiment, the invention provides cells that can have theability, or the enhanced ability, to transport or produce substrates.These cells can have nucleic acid sequences that encode polypetides withtransporter activity (e.g., itr1 from S. cerevisiae) and/orinositol-1-phophate synthase activity as described herein.

In another embodiment, the invention provides cells having reducedL-gulonate 3-dehydrogenase activity (E.C. 1.1.1.45). Such cells also cancontain a polypeptide having phytase activity, a polypeptide havingphosphatase activity (or a mixture of polypeptides having differentphosphatase activities), and/or a mixture of polypeptides having phytaseactivity and polypeptides having phosphatase activity (or polypeptideshaving different phosphatase activities). In addition, such cells cancontain exogenous nucleic acid molecules that encode a polypeptidehaving phytase activity, a polypeptide having phosphatase activity (or amixture of polypeptides having different phosphatase activities), and/ora mixture of polypeptides having phytase activity and polypeptideshaving phosphatase activity (or polypeptides having differentphosphatase activities). For example, a cell can contain polypeptideshaving multiple inositol polyphosphate phosphatase activities orexogenous nucleic acid molecules that encode polypeptides havingmultiple inositol polyphosphate phosphatase activities. Cells havingphytase activity, phosphatase activity, and/or mixtures thereof as wellas reduced L-gulonate 3-dehydrogenase activity can be used to produceincreased levels of myo-inositol from phytic acid.

8. Organic Compound Production and Culturing Methods

The invention provides methods for producing an organic compound. Forexample, the methods and materials described herein can be used toproduce D-glucose, D-glucose-1-phosphate, D-glucose-6-phosphate,UDP-D-glucose, D-myo-inositol, D-myo-inositol-1-phosphate,D-glucuronate, D-glucuronate-1-phosphate, UDP-D-glucuronate,D-glucurono-3,6-lactone, L-gulonate, L-gulono-γ-lactone, glucaric acid,and L-ascorbate. Other examples of compounds that can be producedinclude, without limitation, L-dehydroascorbate, L-threonate, and3-dehydro-L-threonate. It is noted that a produced compound can be inthe D or L configuration. In addition, a polypeptide having a particularenzymatic activity can be used such that the desired organic compound isoptically pure (e.g., about 75, 80, 85, 90, 95, 99, or 99.9 percentpure).

A cell described herein can be used to produce a particular organiccompound such as myo-inositol, ascorbic acid, or glucaric acid. Forexample, a microorganism containing all the polypeptides necessary toproduce ascorbic acid from glucose as depicted in FIG. 1 can be used toproduce ascorbic acid. Alternatively, different microorganisms can beused to produce a particular organic compound. For example, threedifferent microorganisms each containing a different set of polypeptidesnecessary to produce ascorbic acid from glucose can be used to produceascorbic acid. In other words, one or more than one group of cells canbe used to produce a particular organic compound.

In addition, purified polypeptides having enzymatic activity can be usedalone or in combination with cells to produce organic compounds. Forexample, with reference to FIG. 1, a microorganism containingpolypeptides necessary to catalyze steps one through six can be used toproduce L-gulono-γ-lactone from glucose, while a purified polypeptidehaving gulono-γ-lactone oxidase activity (EC 1.1.3.8) can be used toconvert L-gulono-γ-lactone into L-ascorbate. Any method can be used topurify a particular polypeptide. For example, size fractionation, ionexchange, HPLC, and affinity chromatography can be used to purify apolypeptide having enzymatic activity. In addition, purifiedpolypeptides can be used on a solid support (e.g., glass beads, polymerstructures, and other plastics), or in solution.

Further, cell free extracts containing a polypeptide having enzymaticactivity can be used alone or in combination with purified polypeptidesand/or cells to produce organic compounds. For example, with referenceto FIG. 1, a microorganism containing polypeptides necessary to catalyzesteps one through five can be used to produce L-gulonate from glucose,while a cell-free extract containing a polypeptide having 1,4-lactonehydroxyacylhydrolase activity (EC 3.1.1.25) is used to convertL-gulonate into L-gulono-γ-lactone, and a purified polypeptide havinggulono-γ-lactone oxidase activity (EC 1.1.3.8) is used to convertL-gulono-γ-lactone into L-ascorbate. Any method can be used to produce acell-free extract. For example, French pressure cell disruption,enzymatic lysis, mechanical shearing (with, for example, glass beads),osmotic shock, sonication, and/or a repeated freeze-thaw cycle followedby filtration and/or centrifugation can be used to produce a cell-freeextract from intact cells.

It is noted that a cell, purified polypeptide, and/or cell-free extractcan be used to produce a particular organic compound that is, in turn,treated chemically to produce another organic compound. For example, amicroorganism can be used to produce L-gulono-γ-lactone, while achemical process is used to convert L-gulono-γ-lactone into L-ascorbate.Such chemical processes include, without limitation, treatment withbenzaldehyde-hydrogen chloride, oxidation with manganese dioxide, andhydrolysis with 70 percent acetic acid-water (Crawford and Crawford,Adv. in Carbohydrate Chem. Biochem., 37:79-155 (1980)). Likewise, achemical process can be used to produce a particular organic compoundthat is, in turn, converted into another organic compound using amicroorganism, purified polypeptide, and/or cell-free extract describedherein. For example, a chemical process can be used to produceL-gulono-γ-lactone, while a microorganism is used to convertL-gulono-γ-lactone into L-ascorbate.

Typically, a particular organic compound is produced by providing cellsand culturing the provided cells with culture medium such that thatorganic compound is produced. In general, the culture media and/orculture conditions can be such that the cells grow to an adequatedensity and produce the desired compound efficiently.

For large-scale production processes, the following methods can be used.First, a large tank (e.g., a 50-, 100-, 200-, or more gallon tank)containing appropriate culture medium with, for example, hexose and/orpentose carbons is inoculated with a culture of a particular cell. Afterinoculation, the cells are incubated to allow the production of biomass.Once a sufficient biomass is reached, the broth containing the cells canbe transferred to a second tank. This second tank can be any size. Forexample, the second tank can be larger, smaller, or the same size as thefirst tank. Typically, the second tank is larger than the first suchthat additional culture medium can be added to the broth from the firsttank. In addition, the culture medium within this second tank can be thesame as, or different from, that used in the first tank. For example,the first tank can contain medium with xylose and arabinose, while thesecond tank contains medium with glucose.

Once transferred, the cells are incubated to allow for the production ofthe desired organic compound. Once produced, any method can be used toisolate the desired compound. For example, common separation techniquescan be used to remove the biomass from the broth, and common isolationprocedures (e.g., extraction, distillation, and ion-exchange procedures)can be used to obtain the organic compound from the cell-free broth. Inaddition, the desired organic compound can be isolated while it is beingproduced, or it can be isolated from the broth after the productproduction phase has been terminated.

It will be appreciated that the methods and materials described hereincan be adapted and used in any type of culturing process including,without limitation, the processes commonly referred to as “continuousfermentation” and “batch fermentation” processes. In addition, the cellsused during one production process can be recovered and reused insubsequent production processes. For example, the cells can be reusedmultiple times to produce a desired organic compound. Further, anycarbon source can be used. For example, allose, altrose, glucose,mannose, gulose, iodose, galactose, talose, melibiose, phytic acid,sucrose, fructose, raffinose, stachyose, ribose, arabinose, xylose,lyxose, glycerol, inositol, carbon combinations such as inositol andglucose, starches such as molasses, corn starch, and wheat starch, andhydrolysates such as corn fiber hydrolysates and other cellulosichydrolysates can be used as a carbon source for producing either biomassor the desired organic compound. Moreover, any medium can be used. Forexample, standard culture media (e.g., yeast minimal medium and YPmedium (yeast extract 10 g/L, peptone broth 20 g/L)) as well as mediasuch as corn steep water and corn steep liquor can be used.

Phytic acid can be converted into myo-inositol, which is then convertedinto ascorbic acid as described herein. Any method can be used toproduce ascorbic acid from phytic acid. For example, chemical methodscan be used to convert phytic acid into myo-inositol, while enzymaticmethods are used to convert myo-inositol into ascorbic acid. Anymaterial containing phytic acid such as corn steep liquor can be used asa source material. In addition, phytic acid can be used in a pure orunpure form. In one embodiment, phytate is purified from a solution suchas corn steep liquor and converted into inositol by chemical hydrolysis.In this case, the resulting inositol can be enzymatically converted intoascorbic acid.

Any method can be used to purify phytate from a solution. For example,calcium phytate can be recovered from materials such as corn steepliquor, heavy steep water, or light steep water by treating the liquidmedium with a calcium compound such as calcium hydroxide (e.g., a 15percent solution of calcium hydroxide). After treatment, the pH can beadjusted to about 6.0. Once formed, the calcium phytate product can bewashed with warm water (e.g., 50° C. water) and filtered to removeimpurities. This process can yield insoluble calcium phytate that can befurther converted to inositol.

Alternatively, a solution containing phytate can be treated with an ionexchange resin such as that described in U.S. Pat. No. 4,668,813. Afteradsorbtion of the phytate to the resin, the resin can be washed withwarm water (e.g., 30° C. to 85° C. water), and the phytate eluted as asalt by treating the bound resin with a solution such as aqueous sodiumhydroxide, potassium hydroxide, ammonium hydroxide, or the like. Anothermethod that can be used to isolate phytic acid involves separatingphytate from steep water as described in U.S. Pat. No. 3,410,929.Briefly, steep water can be passed over a resin such as Dow ChemicalCompany Retardation 11 A8 resin. After passage of the steep water, theresin can be washed with water, and the phytate desorbed from the resinby washing with a NaCl solution.

Any method can be used to convert phytic acid into inositol. Forexample, inositol can be made from phytic acid by treating phytic acidin water at 100° C. as described elsewhere (Cosgrove, D. J. “InositolPhosphates” Elsevier, Amsterdam, 1980, p. 36). Alternatively, inositolcan be derived from phytate by steam treatment as described elsewhere(e.g., U.S. Pat. No. 4,668,813) or by enzymatic treatment as describedelsewhere (e.g., U.S. Pat. No. 5,830,732). In addition, a combination ofenzymatic activities can be used to convert phytic acid into inositol.For example, phytase enzymes can be used to convert phytic acid intoinositol mono-phosphate, and an acid phosphatase can be used to convertthe resulting inositol monophosphate into inositol.

Ascorbic acid can be produced from phytate or inositol using the methodsand materials described herein. For example, a cell (e.g., Saccharomycescerevisiae) expressing polypeptides having (1) phytase activity capableof converting phytate into inositol or phytase and acid phosphataseactivities, (2) myo-inositol oxygenase activity, (3) glucuronatereductase activity, (4) uronolactonase activity, 1,4-lactonehydroxyacylhydrolase activity, or D-glucono-1,5-lactone lactonohydrolaseactivity, and (5) gulono-γ-lactone oxidase activity, gulono-γ-lactonedehydrogenase activity, or galactono-7-lactone oxidase activity can beused to convert phytate into ascorbic acid by culturing themicroorganism with media containing a high percentage of, for example,corn steep liquor (e.g., a media with 50, 60, 70, 80, 90, 95, or morepercent corn steep liquor). Such a media can contain corn steep liquor(10 g/L, dry basis), ammonium sulfate (3 g/L), biotin (0.1 g/L), andglucose (20 g/L). Alternatively, a cell containing polypeptides havingmyo-inositol oxygenase, glucuronate reductase, uronolactonase, andgulono-γ-lactone oxidase can be used to produce ascorbic acid frominositol. Once produced, any method can be used to purify the resultingascorbic acid. For example, the methods and materials described in U.S.Pat. Nos. 6,037,480 or 6,169,187 can be used to purify ascorbic acidfrom a fermentation broth. Alternatively, the unpurified ascorbic acidcan be used directly as a feed supplement.

9. Modifying Plants and Plant Cells

The invention provides methods and materials related to the use ofplants and plant cells to produce (1) a polypeptide having myo-inositoloxygenase activity, (2) myo-inositol, and/or (3) ascorbic acid.Expression vectors and methods of transforming plant cells are providedherein. These vectors can be designed such that a transgene encoding apolypeptide having myo-inositol oxygenase activity is overexpressed in atransgenic plant and/or plant cell. In one embodiment, the plant orplant cell can be used to produce a polypeptide having myo-inositoloxygenase activity, which in turn can be purified and used in in vitroapplications such as in the production of ascorbic acid.

Plant cells and/or transgenic plants also can be generated as describedherein such that the resulting plant cells and/or transgenic plants haveincreased or decreased responses to environmental stresses. For example,transgenic plant cells or plants can display an increased or decreasedsalt tolerance (Nelson et al., Plant Physiology, 119:165-172 (1999); andNelson, The Plant Cell 10:753-764 (1998)). The modulation of inositolconcentrations also can be useful for altering seed development (Yoshidaet al. Plant Physiology 119:65-72 (1999)). The expression of apolypeptide having myo-inositol oxygenase activity can be increased in aplant cell and/or a transgenic plant by transforming the plant with aconstruct that contains a nucleic acid sequence that encodes apolypeptide having myo-inositol oxygenase activity operably linked to apromoter. Similarly, myo-inositol oxygenase activity can be reduced bytransforming a plant or plant cell with a construct that contains anantisense or sense sequence (Napoli et al., The Plant Cell 2:279-289(1990) and U.S. Pat. No. 5,034,323) which causes the down regulation ofendogenous myo-inositol oxygenase expression. Constructs that eitherup-regulate myo-inositol oxygenase expression or down-regulatemyo-inositol oxygenase expression are herein after termed modulatingconstructs.

Once a nucleic acid sequence encoding a polypeptide having myo-inositoloxygenase activity has been produced, standard techniques can be used toexpress the sequence in transgenic plants. The basic approach is toclone the nucleic acid sequence into a transformation vector such thatit is operably linked to one or more control sequences (e.g., apromoter) that direct expression of the nucleic acid sequence in plantcells. The transformation vector is then introduced into plant cells byone of a number of techniques (e.g., biolistics), whole plants areregenerated from the cells, and progeny plants containing the introducednucleic acid sequence are selected. All or part of the transformationvector can be stably integrated into the genome of the plant cell. Thatpart of the transformation vector which integrates into the plant celland which contains the introduced sequence and associated sequences forcontrolling expression (the introduced (“transgene”) may be referred toas the recombinant expression cassette.

Selection of progeny plants containing the introduced transgene can bemade based upon the detection of an altered phenotype. Such a phenotypemay be enhanced resistance to a chemical agent (such as an antibiotic)as a result of the inclusion of a dominant selectable marker geneincorporated into the transformation vector.

Successful examples of the modification of plant characteristics bytransformation with cloned nucleic acid sequences are replete in thetechnical and scientific literature. Selected examples, which serve toillustrate the knowledge in this field of technology include U.S. Pat.Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615;5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369; and5,610,042. These examples include descriptions of transformation vectorselection, transformation techniques, and the construction of constructsdesigned to over-express the introduced transgene.

The modulating construct can be introduced into a wide variety of plantspecies. These plants can be monocots, dicots, or gymnosperms. Thus, forexample, a nucleic acid encoding a polypeptide having myo-inositoloxygenase activity can be introduced into plant species including,without limitation, maize, wheat, rice, barley, soybean, cotton, beansin general, rape/canola, alfalfa, flax, sunflower, safflower, brassica,cotton, tobacco, flax, peanut, clover, cowpea, grapes; vegetables suchas lettuce, tomato, cucurbits, cassaya, potato, carrot, radish, pea,lentils, cabbage, cauliflower, broccoli, Brussels sprouts, peppers; treefruits such as citrus, apples, pears, peaches, apricots, walnuts; firtrees such as Douglas fir and loblolly pine, and flowers such ascarnations and roses.

A number of recombinant vectors suitable for stable transfection ofplant cells or for the establishment of transgenic plants have beendescribed including those described in Pouwels et al. (Cloning Vectors;A Laboratory Manual, 1985, supp., 1987); Weissbach and Weissbach(Methods for Plant Molecular Biology, Academic Press, 5:173-184, 1989);and Gelvin et al. (Plant Molecular Biology Manual, Kluwer AcademicPublishers, 1990). Typically, plant transformation vectors include oneor more cloned sequences under the transcriptional control of 5′ and 3′regulatory sequences and a dominant selectable marker. Such planttransformation vectors typically also contain a promoter regulatoryregion (e.g., a regulatory region controlling inducible or constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, and RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal. Examples ofconstitutive plant promoters that can be used for expressing a transgeneinclude: the cauliflower mosaic virus (CaMV) 35S promoter, which confersconstitutive, high-level expression in most plant tissues (See, e.g.,Odel et al., Nature, 313:810 (1985); Dekeyser et al., Plant Cell, 2:591(1990); Terada and Shimanoto, Mol. Gen. Genet. 220;389 (1990); andBenfey and Chua, Science, 250:959-966 (1990)); the nopaline synthasepromoter (An et al., Plant Physiol. 88:547 (1988)); the octopinesynthase promoter (Fromm et al., Plant Cell, 1:977 (1989)); and the 2×CaMN/35S promoter with a translation enhance sequence (Kay et al.,Science, 236:1299-1302 (1987)).

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals also canbe used for expression of transgene in plant cells, including promotersregulated by: (a) heat (Callis et al., Plant Physiol., 88:965 (1988);Ainley et al., Plant Mol. Biol., 22:13-23 (1993); and Gilmartin et al.,The Plant Cell., 4:839-949 (1992)); (b) light (e.g., the pea rbcS-3Apromoter, Kuhlemeier et al., Plant Cell, 1:471 (1989), and the maizerbcS promoter, Schaffner & Sheen, Plant Cell, 3:997 (1991)); (c)hormones, such as abscisic acid (Marcotte et al., Plant Cell, 1:471(1989)); (d) wounding (e.g., the potato PinII promoter (Keil et al.,Nucl. Acids. Res. 14:5641-5650 (1986)), the Agrobacterium mas promoter(Langridge et al., Bio/Technology 10:305-308 (1989)), and the grapevinevst1 promoter (Weise et al., Plant Mol. Biol., 26:667-677 (1994)); and(e) chemicals such as methyl jasmonate or salicylic acid (Gatz et al.Plant Mol. Biol. 48:89-108 (1997)).

Alternatively, tissue specific (root, leaf, flower, and seed forexample) promoters (Carpenter et al., The Plant Cell 4:557-571 (1992);Denis et al., Plant Physiol 101: 1295-1304 (1993); Opperman et al.,Science 263:221-223 (1993); Stockhause et al., The Plant Cell 9:479-489(1997); Roshal et al., The EMBO J. 6:1155 (1987); Schernathaner et al.,EMBO J. 7:1249 (1988); Yamamoto et al., Plant Cell 3:371-382 (1990); andBustos et al., Plant Cell 1:839 (1989)) can be fused to the codingsequence to obtain particular expression in respective organs.

Plant transformation vectors also can include RNA processing signals(e.g., introns) that can be positioned upstream or downstream of the ORFsequence in the transgene. In addition, the expression vectors also caninclude additional regulatory sequences from the 3′-untranslated regionof plant genes, e.g., a 3′ terminator region to increase mRNA stabilityof the mRNA, such as the PI-II terminator region of potato or theoctopine of noplaine synthase (NOS) 3′ terminator regions.

Plant transformation vectors also can include dominant selectable markergenes to allow for the ready selection of transformants. Such genesinclude those encoding antibiotic resistance genes (e.g., resistance tohygromycin, kanamycin, bleomycin, G418, streptomycin, or spectinomycin)and herbicide resistance genes (e.g., phosphinothricinacetyltransferase).

Transformation and regeneration of both monocotyledonous anddicotyledonous plant cells is now routine, and the appropriatetransformation technique can be determined by the practitioner. Thechoice of method will vary with the type of plant to be transformed;those skilled in the art will recognize the suitability of particularmethods for given plant types. Suitable methods can include, withoutlimitation, electroporation of plant protoplasts, lipsome-mediatedtransformation, polyethylene glycol (PEG) mediated transformation,transformation using viruses, micro-injection of plant cells,micro-projectile bombardment of plant cells, vacuum infiltration, andAgrobacterium tumefaciens (AT) mediated transformation. Typicalprocedures for transforming and regenerating plants are described in thepatents referenced above.

Following transformation and regeneration of plants with thetransformation vector, transformed plants are usually selected using adominant selectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic resistance on theseedlings of transformed plants, and selection of transformants can beaccomplished by exposing the seedlings to appropriate concentrations ofthe antibiotic.

10. Therapeutic Uses

Polypeptides having myo-inositol oxygenase activity can convertmyo-inositol into glucuronic acid in many eukaryotic organisms,including plants, mammals (e.g., humans), and yeast. This enzymeactivity has been found to be abnormal in kidneys from diabetic animals,and excessive amounts of inositol is secreted in the urine. Abnormalinositol levels are also associated with a number of other clinicalabnormalities (Table I).

TABLE I Affected Condition Tissue Causative Agent(s) Cataract eye lensLow levels of inositol, which is an antioxidant and scavenges excessglucose (diabetes side effect) Adipocyte Adipocyte Incorrect levels ofinositol which is malfunction cells important in membranes and signaltransduction pathways Increased platelet Platelet Increaseduptake/levels of myo- aggregation/ cells inositol and inositolphosphates secretion (diabetes side effect) Hepatic Brain Myo-inositoldepletion in encephalopathy; neurochemical pathways Protacaval shunts;Alzheimer's Nerve conduction Peripheral Motor nerve conduction velocitynerves impaired by low inositol levels (diabetes side effect) Vasculardisease Endothelial Decreased myo-inositol metabolism, cells, decreasedinositol uptake (diabetes neuro- side effect) blastoma Dysmorphogenesis;Mammals Reduced myo-inositol levels Growth retardation (diabetes sideeffect) Immune response B/T Incorrect inositol levels lympho- cytesAtaxia Fibro- Autosomal recessive disorder, telangiectasia blastsaccompanied by alteration in myo- inositol metabolism Renal FailureKidney Infection, trauma, etc.

The identification of a polypeptide having myo-inositol oxygenaseactivity allows for the use of the enzyme and, variants thereof, intherapeutic applications in which abnormal levels of inositol aredetected (see above). Abnormal inositol levels are characterized aslevels of inositol that fall outside of range of inositol levels thatwould be expected from a control group. Abnormal inositol levels couldbe either greater than or less than those displayed by a control group.In many cases the control group can be a random sampling from a normalhealthy population (i.e. a population that does not display outwardmanifestations of a disease that is suspected of being associated withabnormal inositol levels).

As described herein, antisense technology and myo-inositol oxygenasebinding agents can be used to reduce myo-inositol oxygenase activity,and nucleic acid encoding a polypeptide having myo-inositol oxygenaseactivity can be delivered as a therapeutic in cases were increasedmyo-inositol oxygenase activity is desired.

A polypeptide having myo-inositol oxygenase activity, or variantthereof, can be incorporated into a pharmaceutical composition. Foradministration to animals, purified myo-inositol oxygenase polypeptideor variants thereof are generally combined with a pharmaceuticallyacceptable carrier. Pharmaceutical preparations can contain only onetype of myo-inositol oxygenase polypeptide or they can contain acombination of various myo-inositol oxygenase polypeptides. In general,the nature of the carrier will depend on the particular mode ofadministration being employed. For instance, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balance salt, solutions, aqueous dextrose, glycerol, human albumin, orthe like as a vehicle. For solid compositions (e.g., powder, pill,tablet, or capsule forms) conventional non-toxic solid carriers caninclude, for example, pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. In addition to biologically neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example, sodium acetate or sorbitan monolaurate.

The therapeutic compositions described herein can be administered by anyroute. For example, a composition containing a polypeptide havingmyo-inositol oxygenase activity can be administered subcutaneously or byingestion. In addition, a composition containing a polypeptide havingmyo-inositol oxygenase activity can be formulated in a slow-releasecomposition. Slow-release formulations can be produced by combining thepolypeptide with a biocompatible matrix such as cholesterol. Anotherpossible method of administering a polypeptide pharmaceutical is throughthe use of mini osmonic pumps. As stated above a biocompatible carrieralso can be used in conjunction with this method of delivery.

A polypeptide having myo-inositol oxygenase activity can be delivered tocells by introducing a nucleic acid that encodes that polypeptide suchthat the polypeptide is subsequently translated by the host cell. Thiscan be done, for example, through the use of viral vectors or liposomes.Liposomes also can be used for the delivery of the polypeptide itself.

A polypeptide having myo-inositol oxygenase activity or a nucleic acidencoding such a polypeptide can be delivered in conjunction with othertherapeutic agents. These additional therapeutic agents can be used toenhance the therapeutic effect. Examples of additional therapeuticsinclude, without limitation, hormones, ant-inflammatory agents, andantibiotics.

A pharmaceutical composition described herein can be administered by anymeans that achieve its intended purpose. Amounts and regimens for theadministration of can be determined readily by those with ordinary skillin the clinical art of treating diseases. For use in treating theseconditions, the described polypeptides can be administered in an amounteffective to regulate inositiol levels. The described polypeptides canbe administered to a host in vivo, such as for example, through systemicadministration such as intravenous or intraperitoneal administration.Also, the described polypeptides can be administered intralesionally(i.e., injected directly into the affected area).

Effective doses of a myo-inositol oxygenase-based therapeutic treatmentwill vary depending on the nature and severity of the condition to betreated, the age and condition of the subject, and other clinicalfactors. Thus, the final determination of the appropriate treatmentregimen can be made by the attending clinician. Typically, the doserange will be from about 0.1 μg/kg body weight to about 100 mg/kg bodyweight. Other suitable ranges include, without limitation, doses of fromabout 1 μg/kg to 10 μg/kg body weight. The dosing schedule can vary fromonce a week to daily depending on a number of clinical factors such asthe subject's sensitivity to the treatment. Examples of dosing schedulesare 3 μg/kg administered twice a week, three times a week, or daily; adose of 7 μg/kg twice a week, three times a week, or daily; a dose of 10μg/kg twice a week, three times a week, or daily; or a dose of 30 μg/kgtwice a week, three times a week, or daily. In the case of a moreaggressive disease, it may be preferable to administer doses such asthose described above by alternate routes including intravenously orintrathecally. Continuous infusion also can be used.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Cloning Nucleic Acid that Encodes a PolypeptideHaving Myo-Inositol Oxygenase Activity 1. Overview

A polypeptide having myo-inositol oxygenase (MIO) activity fromCryptococcus terreus (ATCC #11799) was partially purified using ionexchange chromatography. This partially pure preparation was furtherpurified by 1-dimensional or 2-dimensional SDS-PAGE. The band and spotscorresponding to likely candidates were carefully excised and subjectedto in situ digestion for peptide separation (μLC/MS/MS) and sequenceanalysis. Both analyses revealed sequences that correlated with anexpressed sequence tag (EST) of a hypothetical polypeptide fromPleurotus ostreatus having unknown function (Accession Number 6934670).Using the entire P. ostrealus EST sequence, BLASTN and BLASTX searchesof GenBank® revealed that putative homologs exist in a wide range oforganisms. One match with the P. ostreatus sequence was the Homo sapiensORF designated EMBL: CAB63064.1 or EMBL: AL096767. Alignments with othertranslated sequences with a high degree of similarity to the H. sapienssequence are presented (FIG. 7).

BLAST searches using the P. ostreatus and Homo sapiens translatedsequences as well as an amino terminal amino acid sequence of a Cr.neoformans polypeptide fragment (C. J. Rosario, MS thesis, CaliforniaState University, Long Beach) against the Cr. neoformans strain H99 ESTsequence database at the University of Oklahoma yielded EST sequencescontaining 5′ and 3′ regions of a cDNA sequence with some similarities.Primers designed from these regions were used to isolate the followingfull-length cDNA sequence from Cr. neoformans strain B3501 (SEQ IDNO:1):

  1 atgcacgctc ccgaagtcaa cgactacatc aagcacaagg ctgttaagct  51cgaccaggtt tctgacgaaa tcgacgaggt caatgtcttg aagctcaagc 101 agaaggacgctgtcgagaag actcaagctg agatcgatta cgacctcgcg 151 agcaagtttg accaagagaaggacaaggct gctttcaggc agtacgagga 201 agcttgcgac cgtgtcaaga acttctacgctgagcaacac ttgaagcaga 251 cctacgagta caatgtaaag atccgacaag aattccgcaacactgtccgt 301 gctcgcatgt ccatctggga agcaatggag ctcctcgaca atctcgtcga351 cgagtccgac cctgacacct ctgttggaca gattgagcac cttcttcaga 401ccgctgaggc tattcgacga gacggcaagc ctgaatggat gcaagtcacc 451 ggtttgattcacgatcttgg caagcttctc tgtttcttcg gtgccgacgg 501 tcagtgggac gttgtcggtgacaccttcgt tgtcggctgc aagttttccg 551 acaagattat ctaccccgac accttcaagtctaaccccga ctataacaac 601 cccaagttga acaccaagta cggtgtctat gagcctaactgcggtttgga 651 caacgtcttg ctcagctggg gtcacgatga gtacatgtac gagatctgca701 agaaccaatc tactcttccc caagaagctc ttgctatgat ccgatatcac 751tctttctacc cctggcaccg agagggtgcc tacgagcatc tcatgaacga 801 gaaggactactcacagctca aggctgtcaa ggctttcaac ccctacgacc 851 tctattccaa gtctgacgacccccccaaga aggaggagct caagccttac 901 taccaaagcc tcatctccaa attcttccctgaggaggtgc agtggtag

2. Methods

Polypeptide concentrations were estimated using the BioRad Protein Assayand the manufacturer's microassay protocol. Bovine gamma globulin wasused for the standard curve determination. This assay is based on theBradford dye-binding procedure (Bradford, M., Anal. Biochem., 72, 248(1976)). Polypeptide content in chromatography fractions was estimatedby absorbance measurements at 280 nm.

Electrophoresis was carried out using a Bio-Rad Protean 3 minigel systemand pre-cast SDS-PAGE gels (4-15% and 12%) or a Protean 3 xi system and16 cm×20 cm×1 mm SDS-PAGE gels (12.5%) cast following the manufacturer'sprotocol. The gels were run according to the manufacturer's instructionswith a running buffer of 25 mM Tris-HCl (pH 8.3), 192 mM glycine, and0.1% SDS (Laemmli buffer system; Laemmli, Nature, 22, 680 (1970)).

Affinity chromatography was performed using Sepharose 6B substitutedwith myo-inositol. Myo-inositol was covalently bound to epoxy-activatedSepharose 6B packing material (Amersham Pharmacia Biotech) following themanufacturer's instructions. Briefly, 2 g of lyophilized epoxy-activatedSepharose 6B was swollen in water for 15 minutes and repeatedly washedwith water on a sintered glass filter. The swollen and washed packingmaterial was suspended in 10 mL of 10 mM NaOH containing 0.4 Mmyo-inositol. The mixture was incubated for 20 hours at 25° C. withgentle shaking. The excess ligand was removed by washing with 1 L water,and any remaining active groups were blocked by incubation with 1 Methanolamine (pH 8.0) for 4 hours at 40° C. The product was then washedthoroughly with water and three cycles of alternating pH. The low pHwash consisted of 0.1 M acetate buffer (pH 4.0) with 0.5 M NaCl, and thehigh pH buffer consisted of 0.1 M Tris-HCl (pH 8.0) with 0.5 M NaCl.This procedure ensured that no free ligand remains ionically bound tothe immobilized ligand. The final product was suspended in 50 mM TEGbuffer (50 mM Tris-HCl (pH 7.0), 0.5 mM EDTA, 100 mg/L glutathione) andstored at 4° C.

About 400-1000 μg of total polypeptide in 50 μL was prepared for2-dimensional SDS-PAGE analysis. Polypeptide samples were resuspended inSDS boiling buffer and heated at 95° C. for 10 minutes. The SDS boilingbuffer contained 5% SDS, 5% BME, 10% glycerol, and 60 mM Tris-HCl (pH6.8). The two-dimensional electrophoresis was performed according to themethod of O'Farrell (J. Biol. Chem., 250: 4007-4021 (1975)). Isoelectricfocusing was carried out in glass tubes with an inner diameter of 2.0 mmusing 2% pH 4-8 ampholines (Gallard-Schlessinger, Garden City, N.Y.) for9600 volt-hours. The SDS in the polypeptide sample was stripped from thepolypeptides during this electrophoretic step. Fifty ng of an IEFinternal standard (tropomyosin) was added to each sample prior toloading. A pH gradient plot was generated using a surface pH electrodeand used to calculate approximate pI values of the various polypeptides.

After equilibration in SDS sample buffer (10% glycerol, 50 mMdithiothreitol, 2.3% SDS, and 0.0625 M tris (pH 6.8)), each tube gel wassealed to the top of a stacking gel that overlays a 10% acrylamide slabgel (0.75 mm thick). SDS slab gel electrophoresis was carried out forabout 4 hours at 12.5 mA/gel. The following polypeptides were obtainedfrom Sigma Chemical Co. (St. Louis, Mo.) and added as molecular weightstandards to the agarose sealing the tube gel to the slab gel: myosin(220,000), phosphorylase A (94,000), catalase (60,000), actin (43,000),carbonic anhydrase (29,000), and lysozyme (14,000). The gels were driedbetween transparent sheets with the acid end to the left.

After running duplicate gels for each sample, each gel was scanned witha laser densitometer (Model PDSI, Molecular Dynamics Inc, Sunnyvale,Calif.). The scanner was checked prior to scanning for linearity with acalibrated Neutral Density Filter Set (Melles Griot, Irvine, Calif.).The images were analyzed using Phoretix 2D Full software (version 5.01)such that major spots and all changing spots were outlined, quantified,and matched on all gels. In cases where polypeptide spots were missingfrom some gels and present in others, a small area of background wasoutlined appropriately to facilitate matching. The general method ofcomputerized analysis for these pairs included automatic spot findingand quantification, automatic background subtraction (lowest onboundary) and automatic spot matching in conjunction with detailedmanual checking of the spot finding and matching functions. Averagedgels were generated for each sample using Phoretix software.

Spot percentages (equal to spot integrated density (volume)) wereexpressed as a percentage of total density of all spots measured.Differences between matched spots in different samples were calculatedfrom averaged spot percentages using Phoretix software according to thefollowing formula:

Difference=(1−average spot % sample x/average spot % sample ref)(−100)

The molecular weight and approximate pI values for each spot weredetermined from algorithms applied to the reference image.

For polypeptide isolation and sequencing, fresh buffers and stainsolutions were prepared daily for the SDS-PAGE gels. A gel thickness of1 mm was used for 1-dimensional and 0.75 mm for 2-dimensional SDS-PAGE.The gels were stained briefly with Coomassie blue (Bio-Rad catalog#161-0436) and then destained to a clear background. The polypeptideband was excised with no excess unstained gel present. An equal area gelwithout polypeptide was excised as a negative control. The gel sliceswere placed in uncolored microcentrifuge tubes, prewashed with 50%acetonitrile in HPLC-grade water, washed twice with 50% acetonitrile,and placed on dry ice until analyzed.

After in-situ enzymatic digestion of the polypeptide sample withtrypsin, the resulting polypeptide fragments were separated bymicro-capillary reverse-phase HPLC. The HPLC was directly coupled to thenano-electrospray ionization source of a Finnigan LCQ quadrupole iontrap mass spectrometer (μLC/MS/MS). Individual sequence spectra (MS/MS)were acquired on-line at high sensitivity for the multiple polypeptidefragments separated during the chromatographic run. The MS/MS spectra ofthe polypeptide fragments were correlated with known sequences using thealgorithm Sequest developed at the University of Washington (Eng et al.,J. Am. Soc. Mass Spectrom., 5, 976 (1994)) and programs describedelsewhere (Chittum et al., Biochemistry, 37, 10866 (1998)). The resultswere reviewed for consensus with known polypeptides and for manualconfirmation of fidelity.

3. Growth of Cryptococcus terreus and Cell Extract Analysis

Cr. terreus (ATCC 11799) was grown at 30° C. with shaking on a mediumconsisting of yeast nitrogen base (YNB) (6.7 g/L), yeast extract (0.3%),0.1 M sodium phosphate, and either myo-inositol (1%), glucose (1%), orno additional carbon source. Similar growth experiments were also donewith YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone)with either myo-inositol (1%), glucose (1%), or no additional carbonsource. The pellets from 50 mL of overnight cultures were washed andresuspended in 50 mM TEGGP buffer (50 mM Tris-HCl (pH, 7.0), 0.5 mMEDTA, 100 mg/L glutathione, 5% glycerol, and protease inhibitor cocktail(“Complete” cocktail at 1 tablet per 10 mL buffer; Roche MolecularBiochemicals)). The cell extracts were prepared by subjecting the washedcell pellets to 3 passes through a Spectronic French pressure cell(mini-cell) at 19,000 psi followed by centrifugation at 20,000×g for 45minutes. To remove any low molecular weight components of the cellextracts that might interfere with the analyses, the clear supernatants(after centrifugation) were applied to Pharmacia PD-10 columns (9.1 mL)previously equilibrated with 50 mM TEGGP buffer. The same buffer wasused to elute the polypeptides from these disposable gel filtrationcolumns. The eluents were assayed for MIO activity.

MIO activity was determined using a modification of the method describedby Reddy et al. (J. Biol. Chem., 256, 8510 (1981)). Briefly, this assayis based on the reaction of orcinol (5-methyl-1,3-benzenediol) with theMIO enzyme reaction product, D-glucuronic acid. The standard assaymixture contained 50 mM Tris-HCl (pH 8.0), 2.0 mM cysteine, 1.0 mMferrous ammonium sulfate, 60 mM myo-inositol, and appropriate quantitiesof enzyme in a total volume of 0.50 mL. The stock L-cysteine solution(0.10 M) was prepared every two weeks, and the stock ferrous ammoniumsulfate solution (0.50 M) was prepared fresh daily. The enzyme solutionsconsisted of cell extracts prepared as described or chromatographyfractions generated during the partial purification of the enzyme. Priorto initiating the reaction by addition of the substrate myo-inositol(stock solution of 0.50 M, dissolved in 50 mM TEGG), all other reagentswere mixed together by vortexing and incubated for 5 minutes at 30° C.The substrate was then added to initiate the assay. After vortex-mixingthe components, the reaction was carried out in an air atmosphere at 30°C. with shaking at 200 rpm for 10 minutes. The reaction was terminatedby the addition of 75 μL of 20% trichloroacetic acid. After vortexing,the assay mixture was transferred to a 1.5 mL polypropylenemicrocentrifuge tube, and the precipitated polypeptide was separated bycentrifugation at 21,000×g for 3 minutes at 25° C. The supernatant wastransferred to a new 1.5 mL polypropylene microcentrifuge tube, and theD-glucuronic acid concentration was determined by the orcinolcolorimetric assay.

The orcinol colorimetric determination assay was performed as follows.Freshly prepared orcinol reagent (0.6 mL; 0.4% (w/v) orcinol, 0.09%(w/v) ferric trichloride hexahydrate in concentrated HCl) was added to0.3 mL of the clear supernatant. The mixture was vortex-mixed and thenincubated in a boiling water bath for 30 minutes. After cooling to 25°C., the mixture was cleared by centrifugation at 21,000×g for 3 minutes.The supernatant was transferred to a disposable cuvette, and theabsorbance was measured at 660 nm. A standard curve was generated byreplacing the enzyme fraction with D-glucuronic acid (0 to 40 μg/mL) andcarrying out the assay as described above. In a typical experiment thespecific activity (μg glucuronate formed/mg protein/10 minuteincubation) of MIO in Cr. terreus grown with inositol as the carbonsource was 3.9 versus 1.1 when grown on glucose.

All reactions were run in duplicate, and all polypeptide fractions wereassayed with and without added myo-inositol substrate (an equal volumeof TEGG buffer with or without substrate was added to initiate theassay). The average of the absorbance readings of the assay mixturescarried out without substrate was subtracted from the average ofreadings of the assay mixtures containing substrate. The difference inthe values was used to calculate the specific activity as μgD-glucuronic acid formed per mg polypeptide in 10 minutes.

Cell extracts from Cr. terreus were assayed for enriched myo-inositoloxygenase (MIO) activity when the cells were grown on myo-inositol vs.glucose. These cell extracts were analyzed by subtractive computeranalysis of 2-dimensional polyacrylamide electrophoresis gels asdescribed above.

The results of the 2-dimensional SDS-PAGE computer analysis of Cr.terreus 11799 cell extracts revealed that a group of about fourpolypeptides are induced when Cr. terreus cells are grown in thepresence of inositol. These polypeptides were numbered 124, 125, 126 and117. Two or more of these spots could be isozymes of the samepolypeptide since their sizes and pI values were similar. The molecularweights of the four polypeptides ranged from 34 to 36 kDa, and their pIsrange from 5.67 to 5.95.

4. Partial Purification of a Polypeptide from Cr. terreus having MIOActivity

Cr. terreus (ATCC 11799) was grown overnight on YNB medium with inositolas the carbon source (26° C. with shaking, final absorbance at 650 nm ofapproximately 1). All operations subsequent to the growth of the cellswere carried out at 1-4° C. unless stated otherwise. The cells from 1.5L of culture were harvested by centrifugation at 12,000×g for 10 minutesand washed with 2×200 mL of 10 mM TEGG buffer (10 mM Tris-HCl (pH 7.0),0.5 mM EDTA, 100 mg/L glutathione, and 5% glycerol), centrifugingbetween washes to pellet the cells. The washed cells (24.2 g wet cellweight) were re-suspended in 20 mL 10 mM TEGGP buffer containingprotease inhibitor cocktail (“Complete” protease inhibitor cocktailtablets at 1 tablet per 10 mL buffer; Roche Molecular Biochemicals) anddisrupted by three passes through a Spectronic French pressure cell(40K) at 25,000 psi. The cell debris was removed by centrifugation at20,000×g for 45 minutes, and the pellet was discarded. The supernatantwas desalted by separation on Pharmacia disposable PD-10 columns (2.5 mLextract loaded per column) that had each been previously equilibratedwith 20 mL 10 mM TEGG buffer followed by 5 mL 10 mM TEGGP buffer. Thepolypeptide fraction was eluted with 3.5 mL of 10 mM TEGGP buffer percolumn (total volume=42 mL).

The resulting cell extract was applied over three runs to a Bio-Rad UnoQ(6 mL) column previously equilibrated with 10 mM TEGG buffer. The anionexchange column was washed with 30 mL 10 mM TEGG and eluted with alinear gradient of 0 to 400 mM NaCl in 10 mM TEGG buffer (50 mL)followed by a step gradient to 2.0 M NaCl (12 mL). The flow rate for theelution was 1 mL/minute, and 5 mL fractions were collected. Thefractions were assayed using the MIO-orcinol assay procedure describedabove. To achieve accurate activity results, each fraction from the UnoQchromatography was desalted before assaying as described above. Thefractions determined to have MIO activity were pooled.

The active desalted polypeptide fractions were further purified byseparation on an Inositol Sepharose column (1×10 cm) previouslyequilibrated with 10 mM TEGG buffer. The affinity column was washed with25 mL of the same buffer and eluted with a linear gradient of 0 to 200mM NaCl in 10 mM TEGG buffer (24 mL) followed by a step gradient to 500mM NaCl (7 mL). The polypeptide fractions determined to have activityusing the MIO-orcinol assay procedure were pooled and stored at 4° C.The results of a typical preparation, starting with 1.5 L of cellculture are shown in Table II. Specific activity was calculated bydividing the amount of glucuronate produced by the total polypeptide ofthe sample. The relative purity of the M10 polypeptide duringpurification was estimated by the relative intensity of the band onone-dimensional gel electrophoresis.

TABLE II Purification of a polypeptide from Cr. terreus 11799 having MIOactivity Specific Activity Volume [polypeptide] Activity activity/mgPurification Fraction mL mg/mL per mL polypeptide -fold Cell freeextract 17.5 13.95 54.1 3.9 1 Uno Q 14.0 0.48 134.0 278.2 71Inositol-Sepharose 4.0 0.19 134.8 709.5 182 (activity is defined as μgglucuronate formed during assay)

5. Polypeptide Sequencing

A polypeptide sample from Cr. terreus (ATCC # 11799) having MIO activitythat was partially purified using ion exchange chromatography on aBio-Rad UnoQ column (6 mL) was further purified by 1-dimensionalSDS-PAGE using a 16 cm×20 cm×1 mm 12.5% slab gel. The gel was stainedbriefly with Coomassie blue, and a band corresponding to a molecularweight of about 35 kDa was excised and sequences as described above.

Three consensus sequences were identified from three of the MS/MSspectra and manually confirmed to be as follows: DGKPEWMQVTGLVHDLGK (SEQID NO:2); DGKPEWM*QVTGLVHDLGK (SEQ ID NO:3); and YHSFYPWHR (SEQ IDNO:4). These three sequences correlated with a hypothetical polypeptideof unknown function from Pleurotus ostreatus, also known as oystermushroom.

A polypeptide sample from Cr. terreus (ATCC # 11799) having MIO activitythat was partially purified by anion exchange chromatography on aBio-Rad UnoQ column and affinity chromatography using Inositol-Sepharosewas further separated using 2-dimensional electrophoresis. Spots fromthese gels were correlated with spots from the 2-dimensional gels of Cr.terreus cell extracts subjected to limited subtractive computer analysisas described above. One of these spots (#117 of the cell extract gels;MW=35,700 Da; pI=5.95) predominated in the gels of the partiallypurified MIO sample and was chosen for sequence analysis. This spot wasexcised from the gel and treated as described above for in situdigestion, peptide separation (μLC/MS/MS), and sequence analysis. Thefollowing two distinct consensus sequences were identified:DGKPEWMQVTGLVHDLGK (SEQ ID NO:2) and YHSFYPWHR (SEQ ID NO:4).

These sequences are identical to those obtained from the partiallypurified sample separated by 1-dimensional gel electrophoresis.

The data generated from these polypeptide samples revealed sequencesimilarity to partial sequences of an EST clone (gi:6934670) fromPleurotus ostreatus. Using the entire P. ostreatis EST as a template, asearch of the public gene sequence databases was performed to search forputative mio homologs in a wide range of organisms. BLASTN and BLASTXsearches revealed several full-length homologs. The sequence with thehighest similarity to the P. ostreatus EST was a 217 amino acid putativepolypeptide from Pinus radiata (Monterey pine; gi:293552; E score of 7e⁻⁵⁷). The searches also revealed three ORFs from Arabidopsis thaliana(thale cress; gb:AAF43953.1; gb:AAC62136.1; gb: pir: T06010), two ORFsfrom Homo sapiens (human; gb:AAF25204.1; emb: CAB63064.1), and one ORFfrom Rattus norvegicus (rat; gb: AAF25203.1). Other sequences wereidentified in Mus musculus (mouse) as well as in rice and tomato ESTdatabases. The sequence of one of the A. thaliana sequences (gb:AAC62136.1) contained an intron, and the sequence was corrected todelete this region. FIG. 7 is an alignment of several sequences sharingsequence similarity with the Pinus radiata sequence.

6. Nucleic Acid Cloning

BLAST searches using the P. ostreatus and Homo sapiens translatedsequences as well as an amino terminal amino acid sequence of a Cr.neoformans polypeptide fragment (C. J. Rosario, MS thesis, CaliforniaState University, Long Beach) against the Cr. neoformans (strain H99)EST sequence database at the University of Oklahoma were performed. OneEST exhibited sequence similarity with all three sequences including theamino terminal region. By alignment analysis with the 5′ and 3′ regionsof this clone (designated a7e05cn.r1 and a7e05cn.f1), putative start andstop codons were identified. Primers were designed from these sequencesand were used to isolate the entire cDNA sequence from Cr. neoformans.Specifically, the following PCR primers were designed for cloning intopET30a (Novagen) and pYES2 (Invitrogen):5′-GGCCGGTACCATGGACGCTCCCGAAGTCAA-3′ (SEQ ID NO:5; 5′ primer for bothvectors), 5′-CGCCTCGAGCTACCACTGCACCTCCTCAG-3′ (SEQ ID NO:6; 3′ primerfor pET30), and 5′-GCGCTCTAGACTACCACTGCACCTCCTCAG-3′ (SEQ ID NO:7; 3′primer for pYES2). The restriction sites are underlined, and the startand stop codons in bold.

Both the mio insert for pET30a cloning and the vector were digested withNcoI and XhoI before ligation using the Roche Rapid DNA Ligation Kit.The mio insert and pYES2 were digested with KpnI and XbaI and ligatedusing the Roche Rapid DNA Ligation Kit. The enzyme KpnI leaves an intactKozak sequence (yeast ribosome binding site). Part of the Kozak sequencewas engineered to contain a change (from C to G) in the fourth bp of thecoding sequence. The Cr. neoformans mio nucleic acid was amplified froma Cr. neoformans cDNA library (strain B3501; Stratagene catalog #937052)using the following protocol: (1) 94° C. for 5 minutes, (2) 94° C. for30 seconds, (3) 55° C. for 60 seconds, (4) 72° C. for 1.5 minutes, (5)repeat steps 2-4 9 times, (6) 94° C. for 30 seconds, (7) 55° C. for 60seconds, (8) 72° C. for 1.5 minutes (+5 sec/cycle), (9) repeat steps 6-814 times, (10) 94° C. for 30 seconds, (11) 55° C. for 60 seconds, (12)72° C. for 2.75 minutes, (13) repeat steps 10-12 9 times, and (14) 72°C. for 7 minutes. Sequencing was performed with primers complementary toregions of the vectors adjacent to the multiple cloning sites, and thenprimer walking was performed to generate the full double-strandedsequence.

7. MIO Expression in E. coli

Chemically competent E. coli BLR(DE3) cells were transformed with thepET30a vector containing the Cr. neoformans mio sequence following themanufacturer's instructions. Once transformed, the BLR(DE3) cells weregrown in 50 mL 2× YT medium (16 g tryptone, 10 g yeast extract, 5 gsodium chloride) containing 50 μg/mL kanamycin to an OD₆₅₀ of 0.5 at 37°C. After adding 1 mM IPTG (final concentration) to induce polypeptideexpression, the cells were grown for an additional four hours at 30° C.All operations subsequent to the growth of the cells were carried out at1-4° C. unless stated otherwise. The cells were harvested bycentrifugation at 12,000×g for 10 minutes and washed twice with 50 mMTEGG buffer (50 mM Tris-HCl (pH 7.0), 0.5 mM EDTA, 100 mg/L glutathione,and 5% glycerol) centrifuging between washes (12,000×g; 10 minutes) topellet the cells. The washed cells were lysed using Novagen Bug Busterreagent (5 mL reagent per g wet cell weight) containing Novagenbenzonase nuclease (1 μg benzonase nuclease per mL Bug Buster reagent)and Calbiochem Protease Inhibitor Cocktail III (diluted 1:500) accordingto the manufacturer's instructions. The cell debris was removed bycentrifugation at 21,000×g for 20 minutes, and the pellet was discarded.The supernatant (cell extract) was immediately assayed for myo-inositoloxygenase activity as described above. In a typical experiment, the cellextract from cells expressing the Cr. neoformans sequence exhibited aspecific activity of 138 μg glucuronic acid formed per mg total proteinin 10 minutes. A cell extract from induced cells containing PET30a withno insert exhibited a specific activity of 10.7 μg glucuronic acidformed per mg total protein in 10 minutes.

The Cr. neoformans polypeptide also was purified and tested formyo-inositol oxygenase activity. Briefly, a cell extract from a 250 mLliquid culture of BLR(DE3) cells containing the Cr. neoformans miosequence in pET30, grown in 2× YT medium with 50 μg/mL kanamycin andinduced with 1 mM IPTG, was prepared as described above. The Cr.neoformans polypeptide was purified using a Novagen His-Bind Quick 900cartridge as described in the manufacturer's instructions. The eluent(2.5 mL of the total 4.0 mL in Novagen Elute Buffer) was desalted byseparation on a Pharmacia disposable PD-10 column that had beenpreviously equilibrated with 20 mL of 50 mM TEGG buffer followed by 5 mLof 50 mM TEGG buffer containing protease inhibitor cocktail (50 mMTEGGP). The protein fraction was eluted with 3.5 mL of 50 mM TEGGPbuffer and was immediately assayed for myo-inositol oxygenase activityas described above. The total protein in the samples was determinedusing the Bio-Rad Total Protein Reagent following the manufacturer'sdirections. In a typical assay, the desalted eluent exhibited a specificactivity of 203 μg glucuronic acid formed per mg protein in 10 minutes.

8. MIO Expression in S. cerevisiae

Competent S. cerevisiae INVSc1 cells were transformed with the pYES2vector containing the Cr. neoformans mio sequence. Once transformed, thecells were grown in 20 mL SC-uracil medium containing 2% raffinoseovernight at 30° C. with shaking at 200 rpm following the InVitrogenprotocol. Pelleted cells from the overnight culture were used toinoculate a 250 mL liquid culture in SC-uracil medium containing 2%galactose and 1% raffinose to an OD₆₅₀ of 0.4. The resulting culture wasincubated at 30° C. with shaking. Aliquots were withdrawn at 0, 5, and10 hours after induction with galactose, and the cells were harvested bycentrifugation at 12,000×g for 10 minutes. After centrifugation, thecells were washed with 50 mM TEGG buffer (50 mM Tris-HCl (pH 7.0), 1 mMEDTA, 100 mg/L glutathione, and 5% glycerol), pelleted again, and frozenat −80° C. All of the following operations for cell extract preparationwere carried out at 1-4° C. unless stated otherwise. The washed cellswere resuspended in 50 mM TEGG buffer containing proteases inhibitorcocktail (Roche “Complete” Protease Inhibitor Cocktail Tablets at 1tablet per 10 mL buffer; 50 mM TEGGP) and disrupted by three passesthrough a Spectronic French pressure mini-cell at 19,000 psi. The celldebris was removed by centrifugation at 21,000×g for 30 minutes, and thepellet was discarded. The resulting cell extract was immediately assayedfor myo-inositol oxygenase activity. The total protein in the sampleswas determined using the Bio-Rad Total Protein Reagent following themanufacturer's directions.

In a typical experiment, the cell extract from S. cerevisiae cellsharvested 5 hours after induction of polypeptide expression exhibited aspecific activity of 21.1 μg glucuronic acid formed per mg protein in 10minutes, while the specific activity of the cell extract from cellsharvested 10 hours after induction was 12.8 μg. The specific activity ofthe cell extract from cells harvested just prior to induction was 6.7μg.

Example 2 Cloning Nucleic Acid that Encodes a Human Polypeptide havingMyo-Inositol Oxygenase Activity

E. coli DH10B ElectroMAX cells were purchased from Life Technologies,Inc. (catalog #18290-015), and the plasmid pTRC99A was purchased fromAmersham Pharmacia Biotech (catalog #27-5007). The human kidney cDNAlibrary was purchased from Stratagene (catalog #937250). Bacterialgrowth media components were purchased from Difco or Fisher Scientific,and other reagents were of analytical grade or the highest gradecommercially available. For polypeptides, electrophoresis was carriedout using a Bio-Rad Protean II xi cell gel system. For nucleic acid,electrophoresis was carried out using a Bio-Rad Mini-Sub Cell GT system.An Eppendorf Mastercycler Gradient thermal cycler was used for PCRexperiments. UV-visible spectrometry was done using a Bio-Rad SmartSpec3000 or a Molecular Devices SpectraMAX Plus spectrophotometer.Electroporations were performed using a Bio-Rad Gene Pulser II system.Primers were purchased from Integrated DNA Technologies, Inc. AutomatedDNA sequencing was carried out using an ABI prism 377 DNA sequencer.

PCR primers specific for the Homo sapiens ORF (designated EMBL:CAB63064.1) were designed, and the desired cDNA was amplified from ahuman kidney cDNA library (Stratagene). The primers were as follows:5′-ATATCCATGGAGGTGACGGTGGGCCCAGAC-3′ (SEQ ID NO:8; 5′ primer with NcoIsite) and 5′-CTATTCTAGATCACCAGCTCAGGATGCC-3′ (SEQ ID NO:9; 3′ primerwith XbaI site). The restriction sites are underlined, and the start andstop codons are in bold. The PCR reactions contained 1 μM finalconcentration of each primer; 0.2 mM of dATP, dCTP, dGTP, and dTTP; 2.5units of Expand High Fidelity PCR Polymerase (Roche MolecularBiochemicals); 1 mM MgCl₂; 5 μL of the human kidney cDNA library; and1×HF buffer without MgCl₂ in a 50 μL reaction. The thermocycler programutilized a hot start of 94° C. for 5 minutes; followed by 10 cycles of adenaturing step at 94° C. (30 seconds), an annealing step at 50° C. (1minute), and an extension step at 72° C. (1.5 minutes); 15 cycles of adenaturing step at 94° C. (30 seconds), an annealing step at 50° C. (1minute), and an extension step at 72° C. (1.5 minutes) that increased 5seconds per cycle; 10 cycles of a denaturing step at 94° C. (30seconds), an annealing step at 50° C. (1 minute), and an extension stepat 72° C. (2.75 minutes); and finally a finishing step at 72° C. (7minutes). The amplified DNA with an approximate size of 850 bp waspurified from a 1% agarose gel using a Qiagen QIAquick Gel ExtractionKit and then digested with XbaI and NcoI. The digested DNA was ligatedinto the pTRC99A plasmid (also digested with XbaI and NcoI) at a 5:1molar ratio of insert to plasmid using the Rapid DNA Ligation Kit (RocheMolecular Biochemicals). Transformations into electrocompetent DH10Bwere performed under standard conditions described in the Bio-Radelectroporation manual. Clones containing the human nucleic acid wereidentified by restriction analysis and confirmed by DNA sequencing.

E. coli cells containing the human nucleic acid encoding an enzymehaving MIO activity were grown in 100 mL LB medium containing 100 μg/mLampicillin to an OD₆₅₀ of 0.5 and were induced with 1 mM IPTG (finalconcentration). The induced cells were grown an additional four hours at30° C. All operations subsequent to the growth of the cells were carriedout at 1-4° C. unless stated otherwise. The cells were harvested bycentrifugation at 12,000×g for 10 minutes and washed twice with 50 mMTEGG buffer (50 mM Tris-HCl (pH 7.0), 0.5 mM EDTA, 100 mg/L glutathione,and 5% glycerol), centrifuging between washes (12,000×g; 10 minutes) topellet the cells. The washed cells were re-suspended in 2.0 mL of 50 mMTEGGP buffer containing protease inhibitor cocktail (Roche “Complete”Protease Inhibitor Cocktail Tablets at 1 tablet per 10 mL buffer) andwere lysed with lysozyme (300 μg/mL) for 30 minutes. The cell debris wasremoved by centrifugation at 38,000×g for 45 minutes, and the pellet wasdiscarded. The supernatant was desalted by separation on a Pharmaciadisposable PD-10 column (2.5 mL extract loaded per column) that had beenpreviously equilibrated with 20 mL of 50 mM TEGG buffer followed by 5 mLof 50 mM TEGG buffer containing protease inhibitor cocktail (50 mMTEGGP). The polypeptide fraction was eluted with 3.5 mL of 50 mM TEGGPbuffer and was immediately assayed for myo-inositol oxygenase activityas described in Example 1.

The cell extract from cells containing the nucleic acid encoding thehuman MIO polypeptide exhibited a specific activity of 8.8 μg glucuronicacid formed per mg polypeptide in 10 minutes. A cell extract frominduced cells containing pTRC99A with no insert exhibited a specificactivity of 5.5 μg glucuronic acid formed per mg polypeptide in 10minutes.

Example 3 Activity of Human MIO Polypeptide in Bacillus megaterium Cells

The following PCR primers specific for the Homo sapiens ORF (designatedEMBL: CAB63064.1) are designed, and the desired cDNA is amplified from ahuman kidney cDNA library (Stratagene): 5′ primer with BsrG1 site:5′-ATTATGTACAATGAAGGTGACGGTGGGCCCAGAC-3′ (SEQ ID NO:45) and 3′ primerwith Kpn1 site: 5′-CTATGGTACCTCACCAGCTCAGGATGCC-3′ (SEQ ID NO:46). Thestart and stop codons are in bold, and the restriction sites areunderlined. The PCR conditions are as follows. The reactions contain 1mM final concentration of each primer, 0.2 mM of dATP, dCTP, dGTP, anddTTP; 2.5 units of Expand High Fidelity PCR Polymerase (Roche MolecularBiochemicals); 1 mM MgCl₂; 5 mL of the human kidney cDNA library; and1×HF buffer in a 50 mL reaction. The thermocycler program utilizes a hotstart of 94° C. for 5 minutes followed by 10 cycles of a denaturing stepat 94° C. (30 seconds), an annealing step at 50° C. (1 minute), and anextension step at 72° C. (1.5 minutes); 15 cycles of a denaturing stepat 94° C. (30 seconds), an annealing step at 50° C. (1 minute), and anextension step at 72° C. (1.5 minutes) that increases 5 seconds percycle; 10 cycles of a denaturing step at 94° C. (30 seconds), anannealing step at 50° C. (1 minute), and an extension step at 72° C.(2.75 minutes); and finally a finishing step at 72° C. (7 minutes). Theamplified DNA with a size of about 850 bp is purified from a 1% agarosegel using a Qiagen QIAquick Gel Extraction Kit and then is digested withBsrG1 and KpnI.

A BsrG1 restriction site is inserted into the vector pWH1520 fromMoBiTec LLC by inserting a thymidine (T) nucleotide between bases 4 and5 and by deleting the base at position 8 (A) using the QuickChangeSite-Directed Mutagenesis Kit (Stratagene) following the manufacturer'sprotocol. The resulting plasmid is pWH1520A. The digested PCR product isligated into the pWH1520A plasmid (also digested with BsrG1 and Kpn1)using the Rapid DNA Ligation Kit (Roche Molecular Biochemicals)generating plasmid hsmiopWH1520A. Transformations into electrocompetentDH10B are performed under standard conditions described in the Bio-Radelectroporation manual for this type of cell. Clones containing thehuman mio sequence are identified by restriction analysis and confirmedby DNA sequencing. The hsmiopWH1520A plasmid purified from DH10Btransformants is transformed into commercially available B. megateriumprotoplasts following the manufacturer's protocol (MoBiTec LLC; MarcoIsland, Fla.). Clones containing the hsmiopWH1520A plasmid areidentified by restriction analysis and confirmed by DNA sequencing.

B. megaterium cells containing the hsmiopWH1520A are grown in 100 mL LBmedium containing 10 mg/mL tetracycline to an OD₆₅₀ of 0.3 and areinduced with 0.5% xylose (final concentration). Cells are grown to anOD₆₀₀ of 1.5 and harvested according to the manufacturer's protocol forprotein expression in B. megaterium. All operations subsequent to thegrowth of the cells are carried out at 1-4° C. unless stated otherwise.The cell pellet is resuspended in 50 mM TEGGP buffer (50 mM Tris-HCl (pH7.0), 0.5 mM EDTA, 100 mg/mL glutathione, 5% glycerol, and RocheProtease Inhibitor Cocktail (1 tablet per 10 mL buffer)) and thendisrupted by sonication. After centrifugation at 12,000 rpm for 45minutes to remove the cell debris, the supernatant is desalted byseparation on a Pharmacia disposable PD-10 column (2.5 mL extract loadedper column) that is previously equilibrated with 20 mL of 50 mM TEGGbuffer (50 mM Tris-HCl, 0.5 mM EDTA, 100 mg/mL glutathione, and 5%glycerol) followed by 5 mL of 50 mM TEGGP. The protein fraction iseluted with 3.5 mL of 50 mM TEGGP buffer and is immediately assayed formyo-inositol oxygenase activity.

Example 4 Activity of Human MIO Polypeptide in Yeast Cells

Escherichia coli DH10B ElectroMAX cells were purchased from LifeTechnologies, Inc (catalog #18290-015). Saccharomyces cerevisiae cells(INVSc1; catalog #C810-00) and the pYES2 vector (catalog #V825-20) werepurchased from Invitrogen. The Invitrogen S.c. EasyComp TransformationKit (catalog #K5050-01) was used to prepare and transform INVSc1chemically competent cells. Bacterial growth media components were fromDifco or Fisher Scientific, and other reagents were of analytical gradeor the highest grade commercially available. Plasmids were purified fromE. coli cells using Qiagen Mini and Midi Plasmid Prep Kits, whileplasmids were purified from S. cerevisiae cells using a Zymoprep Kit(Zymo Research; catalog #D2001).

PCR primers specific for the human mio/pTRC99A construct described inExample 2 were designed as follows:5′-AATTGGTACCATGGAGGTGACGGTGGGCCCAGAC-3′ (SEQ ID NO: 10; 5′ primer withKpnI site) and 5′-CTATTCTAGATCACCAGCTCAGG-ATGCC-3′ (SEQ ID NO: 11; 3′primer with XbaI site). The restriction sites are underlined, and thestart and stop codons are in bold. The PCR reactions contained 1 μMfinal concentration of each primer; 0.2 mM of dATP, dCTP, dGTP, anddTTP; 1.75 units of Expand High Fidelity PCR Polymerase (Roche); 1.5 mMMgCl₂, and 0.025 μL of the human mio/pTRC99A plasmid (55 ng/μL) in a 50μL reaction. The thermocycler program utilized a hot start of 94° C. for5 minutes; followed by 10 cycles of a denaturing step at 94° C. (30seconds), an annealing step at 50° C. (1 minute), and an extension stepat 72° C. (1.5 minutes); 15 cycles of a denaturing step at 94° C. (30seconds), an annealing step at 50° C. (1 minute), and an extension stepat 72° C. (1.5 minutes) that increased 5 seconds per cycle; 10 cycles ofa denaturing step at 94° C. (30 seconds), an annealing step at 50° C. (1minute), and an extension step at 72° C. (2.75 minutes); and finally afinishing step at 72° C. (7 minutes). The amplified DNA with anapproximate size of 850 bp was purified from a 1% agarose gel using aQiagen QIAquick Gel Extraction Kit and then digested with KpnI and NcoI.The digested DNA was ligated into the pYES2 plasmid (also digested withKpnI and NcoI) at 4:1 and 6:1 molar ratios of insert to plasmid usingthe Rapid DNA Ligation Kit (Roche Molecular Biochemicals).Transformations into electrocompetent DH10B cells were performed understandard conditions described in the Bio-Rad electroporation manual.Clones containing the human nucleic acid were identified by restrictionanalysis and confirmed by DNA sequencing.

Transformations of competent S. cerevisiae INVSc1 cells with pYES2plasmid containing the human insert (purified from DH10B cells) werecarried out according to the manufacturer's instructions of theInvitrogen S.C. EasyComp™ Transfomation Kit. The transformationreactions were plated on SC minimal plates deficient in uracil(SC-uracil). Clones containing the human nucleic acid were identified byPCR analysis using primers complementary to sequence on both sides ofthe multiple cloning region of pYES2.

Expression of the human MIO polypeptide was induced in & cerevisiaeINVSc1 cells according to the Invitrogen protocol for recombinantproteins in pYES2 under the control of the GAL1 promoter. Cells weregrown in 50 mL SC-uracil medium containing 2% raffinose overnight at 30°C. with shaking at 200 rpm. Pelleted cells from the overnight culturewere used to inoculate a 350 mL liquid culture in SC-uracil mediumcontaining 2% galactose and 1% raffinose to an OD₆₅₀ of 0.4; theresulting culture was incubated at 30° C. with shaking. Aliquots werewithdrawn at 0 and 12 hours after induction with galactose, and thecells were harvested by centrifugation at 12,000×g for 10 minutes. Thepellets were washed with 50 mM TEGG buffer (50 mM Tris-HCl (pH 7.0), 0.5mM EDTA, 100 mg/L glutathione, and 5% glycerol), pelleted again, andfrozen at −80° C.

All of the following operations for cell extract preparation werecarried out at 1-4° C. unless stated otherwise. The cells were disruptedwith glass beads (Sigma, 150-212 microns) in 50 mM TEGGPP buffer (50 mMTris-HCl (pH 7.0), 0.5 mM EDTA, 100 mg/L glutathione, 5% glycerol, Roche“Complete” Protease Inhibitor Cocktail at 1 tablet per 10 mL buffer, and1 mM Pefabloc (Roche Moleuclar Biochemicals)) following the proceduredescribed by Dunn and Wobbe in Current Protocols in Molecular Biologyfor small scale preparations (B. Dunn and C. R. Wobbe, volume 2, section13.13.4, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. F.Seidman, J. A. Smith, K. Struhl eds, John Wiley & Sons, Inc., (1999)).The cell debris and glass beads were removed by centrifugation at21,000×g for 15 minutes. The supernatant was desalted by separation on aPharmacia disposable PD-10 column (2.5 mL extract loaded per column)that had each been previously equilibrated with 20 mL of the 50 mM TEGGbuffer followed by 5 mL of the 50 mM TEGGPP buffer. The polypeptidefractions were eluted with 3.5 mL of the 50 mM TEGGPP buffer and wereimmediately assayed for myo-inositol oxygenase activity as described inExample 1.

In a typical experiment, the cell extract from S. cerevisiae cellsharvested 12 hours after induction of the human MIO polypeptideexhibited a specific activity of 13.4 μg glucuronic acid formed per mgpolypeptide in 10 minutes. A cell extract from similarly induced cellscontaining pYES2 with no insert showed a specific activity of 5.7 μgglucuronic acid formed per mg total protein in 10 minutes.

Example 5 Activity of Human MIO Polypeptide in Insect Cells

The human nucleic acid encoding an MIO enzyme within the pTRC99A plasmiddescribed in Example 2 was subcloned into Baculovirus. Specifically, thepTRC99A-mio clone was digested with Nco I and Sal I, and the 872 bphuman mio fragment was isolated by gel purification. The vectorpFastBacHTa was also digested with Nco I/Sal I, and treated with calfintestinal phosphatase. The human MIO coding sequence was ligated intothe vector in frame with an N-terminal His tag.

DNA sequencing was performed to verify that a polypeptide with thefollowing amino acid sequence was encoded by the construct:

MSYYHHHHHHDYDIPTTEN- LYFQGAMEVTVGPDPSLVYRPDVDPEVAKDKASFRNYTSGPLLDRVFTTYKLMHTHQTVDFVRSKHAQFGGFSYKKMTVMEAVDLLDGLVDESDPDVDFPNSFHAFQTAEGIRKAHPDKDWFHLVGLLHDLGKVLALFGEPQWAVVGDTFPVGCRPQASVVFCDSTFQDNPDLQDPRYSTELGMYQPHCGLDRVLMSWGHDEYMYQVMKFNKFSLPPEAFYMIRFHSFYPWHTGRDYQQLCSQQDLAMLPWVREFNKFDLYTKCPDLPDVDKLRPYYQGLIDKYCPGILSW (SEQ ID NO:12; non-humansequence is underlined, His-Tag is in bold).

The baculovirus transfer vector was used to create bacmid DNA, which wasthen transfected into Sf-9 and Hi5 cells. The virus from thetransfection underwent a single amplification and titering. Theresultant titered primary amplified virus (40 mL volume) was used forthe initial expression screening.

The majority of the 40-mL singularly amplified viral stock was used toinfect 50 mL cultures of Sf-9 and hi5 cells at multiplicities ofinfection (MOI) of 0.1, 1, and 5, each for post infection harvest times(HPI) of 48 hours and 72 hours. In parallel, expressions using the samecells, MOI, and HPI conditions were done with vector alone to serve asnegative controls. Cells and media were harvested for expressionanalysis, and cell extracts were resuspended in TEG buffer (50 mMTris-HCl (pH 7.5), 0.5 mM EDTA, 100 mg/L glutathione) plus 10% glycerol.

Expression analysis investigated the production of soluble polypeptideusing SDS-PAGE and Western blot analysis with His-tag antibodies.Optimal expression was determined to occur in the Hi5 cell line with MOIof 1 or 5, and 48 or 72 hours of post-infection harvest times. Sampleswere flash-frozen with liquid nitrogen and placed on dry ice untilassayed. MIO activity assays were performed as described in Example 1.Hi5 cells expressing the human polypeptide exhibited MIO activity, whileHi5 cells lacking the human polypeptide did not (FIG. 8). Specificactivity in FIG. 8 is defined as μg glucuronate formed/mg protein/10minute incubation.

Further expression experiments were done at a MOI of 1 and a 48-hourHPI. Cell paste was suspended (about 6 g of each clone from 0.5 L insectculture) in 50 mL lysis buffer (1×PBS (pH 7.4), 10% glycerol, 1 μg/mLpepstatin, 5 μg/mL leupeptin, 1 μM E64, 100 mg/L glutathione). Proteaseinhibitors and glutathione were added just before use. The cellsuspension (total volume about 60 mL) was kept on ice and sonicated witha macrotip 2 times (30 seconds each; output=7; duty=70%) with a BransonSonifier 450. The whole cell lysate was centrifuged at 4° C. for 30minutes at 11,000 rpm in a Beckman JA-14 rotor (18,600 g). Thesupernatant was removed (about 60 mL) and loaded onto a 2 mL QiagenNickel-NTA column (previously pre-washed and pre-equilibrated in lysisbuffer) at 4° C. The column was washed with 20 mL wash buffer (lysisbuffer plus 0.17 M NaCl plus 10 mM imidazole) at 4° C. Polypeptides wereeluted with 2.5 mL of elution buffer (wash buffer plus 0.2 M imidazole).The molecular mass and purity of the recombinant polypeptide wasverified by SDS-PAGE for each step in the purification. The eluatepolypeptides were passed through a PD-10 column (Amersham PharmaciaBiotech, Piscataway, N.J.) and eluted in final storage buffer (50 mMTris-HCl (pH 7.5), 0.5 mM EDTA, 100 mg/L glutathione, 10% glycerol).Total polypeptide concentrations were measured using the Bradford assay.Samples were aliquoted into 1 mL volumes, flash-frozen with liquidnitrogen, and placed at −70° C. until analyzed.

The eluate was purified to homogeneity as judged by SDS-PAGE analysis.MIO activity assays were performed on all of the fractions from thepurification process using the methods described in Example 1. Resultsindicated that the purified polypeptide has a specific activity of 225μg glucuronic acid formed per mg polypeptide during a 10 minute assay.

Example 6 Human Cells Containing Exogenous Nucleic Acid that Encodes aHuman Polypeptide Having Myo-Inositol Oxygenase Activity

Human kidney embryonic cells (293 cells; ATCC catalog number CRL 1573)are maintained in DMEM supplemented with 10% fetal calf serum (GIBCO), 2mM glutamine, 100 unit/mL penicillin, and 100 mg/mL streptomycin. Anucleic acid insert containing the human mio gene is introduced into the293 cells using the general procedure described by Caruso et al. (Proc.Natl. Acad. Sci. USA, 93:11302-11306 (1996)). Briefly, a vector isconstructed to contain the human nucleic acid encoding the MIO enzymedescribed in Example 2 using a replication-defective adenoviral vector(ADV) under the transcriptional control of the Rous sarcoma virus longterminal repeat promoter (Accession Number gi:61690). The internalribosome binding site (nucleotides 40-35) is obtained from the pCITE-1vector (Novagen, Madison, Wis.) and is inserted into the E1 deletedadenovirus backbone pAd.1/Rous sarcoma virus as described elsewhere(Fang et al., Gene Ther., 1,:247-254 (1994)). Recombinant adenovirus isgenerated by cotransfection with pBHG10 in 293 cells as described byBett et al. (Proc. Natl. Acad. Sci. USA, 91:8802-8806 (1994)). Titersare calculated using a plaque assay (pfus).

Cells are seeded on a six-well plate (30,000-50,000/cm²) using 0.25%trypsin. About one million cells are infected with the ADV/mio virus atvarious multiplicities of infection (e.g., about 200-1000) in a totalvolume of 0.5 mL. Infection is stopped after a two hour incubation at37° C. by decanting the viral supernatant and adding 2.5 mL fresh cellmedium. Cells are harvested after incubation at 37° C. for 48-72 hoursand resuspended in lysis buffer (1×PBS (pH 7.4), 10% glycerol, 1 μg/mLpepstatin, 5 μg/mL leupeptin, 1 μM E64). The cell suspension issonicated on ice and centrifuged (18,600 g) at 4° C. to remove cellulardebris. The whole cell lysate is assayed for MIO activity as describedin Example 1.

Example 7 Cloning Nucleic Acid that Encodes a Cryptococcus neoformansPolypeptide Having Myo-Inositol Oxygenase Activity into Cryptococcusneoformans

A nucleic acid insert containing the Cr. neoformans mio gene isintroduced into Cr. neoformans using the general procedure described bydelPoeta et al. (Infect. Immun., 67(4):1812-1820 (1999)). The nucleicacid encoding a Cr. neoformans polypeptide having MIO activity isamplified from a Cr. neoformans cDNA library (strain B3501; Stratagenecatalog #937052) using the Expand High Fidelity PCR system (RocheMolecular Biochemicals) and the following PCR primers:5′-CACATCTAGAATGCA-CGCTCCCGAAGTCAA-3′ (SEQ ID NO: 13) and5′-TTAAGGTACCCTACCACTG-CACCTCCTCAG-3′ (SEQ ID NO: 14). Restriction sitesare underlined. The PCR conditions are as follows: (1) 94° C. for 5minutes, (2) 94° C. for 30 seconds, (3) 55° C. for 60 seconds, (4) 72°C. for 1.5 minutes, (5) repeat steps 2-4 9 times, (6) 94° C. for 30seconds, (7) 55° C. for 60 seconds, (8) 72° C. for 1.5 minutes (+5seconds/cycle), (9) repeat steps 6-8 14 times, (10) 94° C. for 30seconds, (11) 55° C. for 60 seconds, (12) 72° C. for 2.75 minutes, (13)repeat steps 10-12 9 times, and (14) 72° C. for 7 minutes. This PCR isused to produce a fragment (about 1 Kbp) that is digested with XbaI andKpnI and inserted into the XbaI/KpnI site of pUC 19 to yield pMIO1.

The GAL7 promoter is amplified from genomic DNA (gDNA) of Cr. neoformansstrain JEC21 (ATCC 96910) using the following PCR primers:5′-GACCAAGCTTGTGGA-AAGAAGCAGGTCTTGTCGA-3′ (SEQ ID NO: 15) and5′-GGCTAAGCTTTCTCAAGAG-GGGATTGAGCGCTGA-3′ (SEQ ID NO: 16). Restrictionsites are underlined. The PCR conditions are as follows: (1) 95° C. for5 minutes, (2) 93° C. for 50 seconds, (3) 50° C. for 50 seconds, (4) 72°C. for 80 seconds, and (5) 72° C. for 2 minutes; with steps 2 through 4being repeated 25 times. This PCR is used to produce a fragment (about585 bp) that is digested with HindIII and inserted into the HindIII siteof pMIO1 to yield pGAL7::MIO.

The Cr. neoformans phosphoribosylaminoimidazole carboxylase (ade2) geneamplified from a Cr. neoformans cDNA library (strain B3501; Stratagenecatalog #937052) is then inserted downstream from the MIO nucleic acidinto the EcoRI site to yield pGAL7::MIO/ADE2. The ade2 gene is isolatedusing the following PCR primers: 5′-AATTGAATTCCCGGTGGACCA-AGTGGAAGC-3′(SEQ ID NO:17) and 5′-AATTGAATTCGCACAGACACCGCCCGTACT-3′ (SEQ ID NO:18).Restriction sites are underlined. The PCR conditions are as follows: (1)95° C. for 5 minutes, (2) 93° C. for 50 seconds, (3) 50° C. for 50seconds, (4) 72° C. for 3 minutes, and (5) 72° C. for 2 minutes; withsteps 2 through 4 being repeated 30 times. This PCR is used to produce afragment (about 2.5 Kbp) that is digested with EcoRI and inserted intothe EcoRI site of pGAL7::MIO to yield pGAL7::MIO/ADE2.

The pGAL7::MIO/ADE2 construct is transformed into Cr. neoformans M001(ATCC MYA-428; an ade2 auxotrophic mutant of strain H99) by biolisticdelivery of DNA as described elsewhere (Toffaletti et al., J.Bacteriol., 175:1405-1411 (1993)).

Adenine prototrophs are selected on synthetic medium (without adenine)supplemented with 1 M sorbitol at 30° C. Synthetic medium consists of(per liter) 6.7 g of yeast nitrogen base without amino acids (YNB w/o),1.3 g of amino acid mix lacking adenine, 180 g of sorbitol, 20 g ofgalactose, and 20 g of agar. Adenine transformants are subcultured ontoselective medium (YNB-galactose) and then are passaged twice on yeastextract-peptone-dextrose (YEPD) agar to select for stable transformants.

Example 8 Enzymatic Activities in Saccharomyces cerevisiae andEscherichia coli Cells

Nucleic acid molecules encoding the following polypeptides were clonedinto Saccharomyces cerevisiae and Escherichia coli cells: (1) Sus scrofaglr (glucuronate reductase; gi: 1703236), (2) Zymomonas mobilis ula(Gluconolactonase; gi:48654), (3) Fusarium oxysporum lha(lactonohydrolase, broad specificity; gi:3810872; AB010465.1), and (4)Rat glo (Gulono-γ-Lactone Oxidase, cDNA; NM_(—)022220).

1. Sources of Template DNA

Fusarium oxysporum ATCC 48112 was grown on a potato sucrose medium and aglycerol corn steep liquor medium to early log, mid-log, and stationaryphases at 26° C. with shaking at 270 rpm. The mycelia were harvested bycentrifugation at 3500 rpm for 10 minutes, washed with 50 mM Tris-HCl,pH 7.0 buffer (cold), and spun again. The resulting washed pellets werequick frozen at −80° C. Using ten grams of mycelia, a custom cDNAlibrary (Stratagene, Inc.; Uni-ZAP XR vector) was synthesized. Theestimated amplified titer of the resulting custom cDNA library was9.6×10⁹ pfu/mL, the average insert size was 1.5 kB, and the insert sizerange was 0.80 to 2.2 kB.

Zymomonas mobilis ATCC 29191 was grown in the recommended ATCC culture,and the gDNA was isolated using the Promega gDNA isolation kit forGram(−) organisms.

A commercial rat liver cDNA library (Clonetech, Inc.) was purchased forglo and glr cloning.

2. Cloning

All constructs were cloned into S. cerevisiae using the shuttle vectorpYES2 (Invitrogen Life Technologies, Carlsbad, Calif.). The constructsequences all included a Kozak sequence (ATCATGG) where the bold lettersdenote the start codon. PCR primers were designed based on the GenBanksequences and included restriction sites for cloning into the multiplecloning site of pYES2. PCR primers for the lha gene from F. oxysporumwere designed based on the sequence of the lactonohydrolase for theproposed mature form in which the first 20 amino acids (leader sequence)have been cleaved (Kobayahi et al, Proc. Natl. Acad. Sci. USA,95(22):12787-12793 (1998)). An ATG start codon was designed into theN-terminal primer preceding the codon GCT, encoding the 21^(st) aminoacid (Ala) of the immature polypeptide. The sequence of these primers isas follows:

(SEQ ID NO:47) N-terminal: 5′-CGCGGATCC ATGGCTAAGCTTCCTTCTACG-3′

The BamHI site is shown in italics, and the start codon is in bold type

(SEQ ID NO:48) C-terminal: 5′-CGACTCGAG CTAATCATAGAGCTTGGGACC-3′

The Xho1 site is shown in italics, and the stop codon is in bold type

Amplification of the rat glo sequence by PCR was performed as describedbelow for the pYES2 cloning using the following reaction mixture:

2 μL rat liver cDNA library (Clontech Catalog #RL5004T)

1 μL of 500 uM PCR primer (each)

4 μL of 10 mM (each) dNTPs

10 μL of 10× Deep Vent polymerase buffer

4 μL of 100 mM MgSO₄

0.5 μL of Deep vent polymerase (2 U/μl)

78.5 μL H₂O

The template, primers, buffer, and MgSO₄ were mixed and heated to 96° C.for minutes. Tubes were then cooled on ice, and the dNTPs and DNApolymerase were added before placing reaction tubes in the thermocycler.The thermocycler protocol was as follows: (1) 96° C. for 3:30, (2) 96°C. for 0:45, (3) 55° C. for 1:15, (4) 72° C. for 4:00, (5) Repeat Steps2-4 34 times, and (6) 72° C. for 10:00.

For all other clones, standard recombinant DNA techniques for PCR,purification of DNA, ligations, and transformations were carried outaccording to established procedures (Sambrook, Fritsch, Maniatis, 1989)or the vendors' protocols. Ligations were typically carried out usingRoche T4 DNA ligase. Initial transformations were typically in E. coliDH10B ElectroMAX cells using the BioRad recommended procedure includingrecovery in SOC medium and plating on LB plates containing ampicillin at100 μg/mL. The purified plasmids (Qiagen miniprep) were screened byrestriction digestion and verified by dideoxynucleotidechain-termination DNA sequencing.

Differences were found in the sequences when compared to the sequenceswithin GenBank®. For the F. oxysporum lha sequence, the followingdifferences were identified: V33V (GTA→GTC); K108K (AAG→AAA); V142V(GTT→GTC); P145P(CCA→CCC); T149T (ACT→ACG); N154N (AAC→AAT); E160E(GAG→GAA); G163G (GGT→GGC); 181T (ACC insert); L184L (CTT→CTC); F189F(TTC→TTT); R191R (CGC→CCT); Q230Q (CAG→CAA); T238T (ACT→ACC); V241V(GTC→GTT); Y304Y (TAT→TAC); and R358R (AGG→AGA). For the Z. mobilis ulasequence, the following differences were identified: T2A (ACC→GCC;intentional from introduction of Kozak sequence); V16A (GTT→GCC); M171(ATG→ATA); 1201 (ATC→ATT); A25A (GCA→GCC); E34Q (GAG→CAG); 1961(ATT→ATC); 163V (CGT insertion); S237S (TCC→TCT); P238P(CCG→CCT); andD261D (GAT→GAC). For the Rat glo sequence, the following differenceswere identified: 185V (ATA→GTA); R168R (AGA→CGA); and Q189H (CAG→CAC).For the Rat glr sequence, the following differences were identified:T→A(ACG→GCG; intentional from introduction of Kozak sequence).

S. cerevisiae INVSc1 competent cells were prepared using an S.c.EasyComp™ Transformation Kit (Invitrogen Life Technologies, Carlsbad,Calif.), and pYES2 constructs were transformed into the INVSC1 competentcells using the same kit. Transformation reactions were plated onselective media (SC-ura), incubated for 2 days at 30° C., and analyzedby colony PCR.

All of the sequences were subcloned into pET30a. Primers were designedwith restriction sites compatible for the multiple cloning site ofpET30a, and the sequences were amplified by PCR using the pYES2 clonesas template and Expand DNA polymerase (Roche).

The ligations and transformations into E. coli DH10B ElectroMAX cellswere carried out as described above for the pYES2 constructs. Afterverification of the sequences by sequence analysis, the pET30aconstructs were transformed into the expression host BLR(DE3) (Novagen)following the manufacturer's protocol. These constructs were verified byrestriction digestion of the purified plasmids. The glo-pET30a constructwas also transformed into Rosetta(DE3) cells (Novagen). This strainsupplies tRNAs under the control of their native promoters for the rarecodons AUA, AGG, AGA, CUA, CCC, and GGA on a compatiblechloramphenicol-resistant plasmid.

Induction experiments with the pET30a/BLR(DE3) clones were carried outin LB medium containing 50 mg/L kanamycin. Induction experiments withthe glo-pET30a/Rosetta(DE3) clone were carried out in LB mediumcontaining 50 mg/L kanamycin and 34 mg/L chloramphenicol. The cultureswere first grown at 37° C. with shaking at 225 rpm to an OD₆₅₀ between0.5 and 0.8, and protein expression was induced by addition of IPTG. Thelha-pET30a-BLR(DE3) culture was induced with 0.1 mM IPTG and incubatedfor 8 hours at 21° C. (to minimize inclusion body formation) followed bycentrifugation at 12,000×g for 10 minutes to harvest the cells. Theula-pET30a-BLR(DE3) and glo-pET30a-Rosetta(DE3) cultures were inducedwith 0.025 mM IPTG and incubated at 30° C. for 4 hours beforeharvesting. The glr-pET30a-BLR(DE3) cultures were induced with 0.1 mMIPTG and incubated at 30° C. for 4 hours before harvesting.

Cell extracts were prepared using Novagen BugBuster reagent (5 mLreagent per 1 g WCW) containing 1 μL of benzonase protease per mLreagent and 5 μL of Calbiochem protease inhibitor set III per mLreagent. The cell suspension was incubated at room temperature for 15minutes with gentle shaking followed by removal of the cell debris bycentrifugation at 21,000×g for 20 minutes. Alternatively, cell extractswere also prepared by 2 passages through a mini-French pressure cell at19,000 psi (Aminco) followed by centrifugation to remove the celldebris. After centrifugation, the supernatant (cell extract) wascarefully removed, and the enzymes were purified by affinitychromatography using His-Bind 900 cartridges (Novagen). After elutionfrom the His-Bind cartridge, each purified protein was desalted bypassage through a Pharmacia disposable PD-10 column previouslyequilibrated with a buffer compatible with the assay buffer. Forexample, the fusion product of lha (His-LHA) was eluted with 100 mMPIPES, pH 7.0. The desalted cell extracts were used for SDS-PAGEanalysis of soluble proteins and for enzyme assays.

GLR was assayed by following the loss of absorbance at 340 nm (loss ofNADPH) using 0.3 mM NADPH, 10 mM glucuronic acid, and variable amount ofenzyme in 100 mM sodium phosphate, pH 6.6 (Hayashi et al., J. Biochem.,95:2223-2232 (1984)). 1-2 micromoles of NADPH were typically consumedper minute per mg protein.

GLO activity was assayed according to the procedure described byNishikimi (Methods in Enzymology, 62:24-30 (1979)). The assay mixturecontained, in 1 mL, 50 mM potassium phosphate buffer, pH 7.5, 2.5 mML-gulono-γ-lactone, 1 mM EDTA, and enzyme. The reaction was initiated bythe addition of either substrate or enzyme, and the mixture wasincubated with shaking in air for 15 minutes at 37° C. The reaction wasstopped by the addition of 0.1 mL 50% trichloroacetic acid, and theprecipitated protein was removed by centrifugation. The product ascorbicacid was detected by the method described in Example 9. Briefly, 0.956mL 2,2′-dipyridyl reagent was added to 0.15 mL of the supernatantsolution, and the reaction mixture was incubated at 25° C. for 15minutes. After 15 minutes, the sample was centrifuged to remove anyprecipitate, and the absorbance was read at 525 nm. The 2,2′-dipyridylreagent contained 0.056 mL ortho-phosphoric acid (85%), 0.75 mL 0.5%2,2′-dipyridyl (prepared in hot H₂O), and 0.15 mL 1% FeCl₃ (in H₂O).Standards of ascorbic acid (1.0 μg to 10 μg) were run in parallel withthe biological samples for a standard curve. One unit is defined as thequantity that catalyzes the formation of 1 nmol of L-ascorbic acid in 1minute under the conditions described above. Specific activity isexpressed as units per mg of protein (μmol/min/mg).

LHA activity was assayed according to the procedure described by Shimizuet al. (Eur. J. Biochem., 209:383-390 (1992)). The standard assaymixture contained 100 mM PIPES-NaOH (pH 7.0), 150 mM pantoyl-γ-lactone,and enzyme in a final volume of 250 μL. After incubation at 30° C. for30 minutes, the reaction was stopped by the addition of 250 μL methanolcontaining 2 mM EDTA (disodium salt). The supernatant obtained uponcentrifugation at 21,000×g for 5 minutes was analyzed by HPLC. One unit(U) of enzyme is defined as the amount catalyzing the hydrolysis of 1μmole pantoyl-γ-lactone per minute under standard assay conditions.Specific activity is defined as the units of enzyme activity of aprotein fraction divided by the protein concentration of the fraction(mmole/min/mg).

All assay samples were filtered through a 0.45 μm filter and dilutedinto the calibration range of 10 μg/mL to 150 μg/mL for analysis byHPLC. Separation was accomplished using a Symmetry® C18 3.5 μm (4.6×75mm) HPLC column from Waters Corporation, Ireland. The HPLC conditionswere as follows: (1) Flow rate: 0.8 mL/min; (2) Column temp: Ambient;(3) Mobile Phase: 13% methanol pH 2.5 with trifluoroacetic acid; and (4)Detection: UV (220 nm. Under these conditions, pantoic acid elutes atabout 3.5 minutes while pantoyl-γ-lactone elutes at about 2.5 minutes.

Protein concentration was estimated using the BioRad Protein Assay andthe manufacturer's microassay protocol. Bovine gamma globulin was usedfor the standard curve determination. This assay is based on theBradford dye-binding procedure.

3. Results

The glo-pET30a, lha-pET30a, and glr-pET30a constructs expressed solublepolypeptides of the predicted molecular weight as judged by SDS-PAGE. Infact, about 5-10% of total protein in each cell extract was theexpressed polypeptide. The ula-pET30a construct did not express asignificant amount of polypeptide with the predicted molecular weight ofthe ULA fusion protein in a soluble form. The enzyme activity resultswere as follows:

Specific Activity gene enzyme (purified protein) GLR glucuronatereductase 1-3 μmol/min/mg GLO gulono-γ-lactone oxidase 0.926 nmol/min/mgLHA uronolactonase 250 nmol/min/mg ULA D-glucono-1,5-lactonelactonohydrolase ND

LHA was also assayed for the ability to catalyze the formation ofgulonic acid from gulono-γ-lactone using the same protocol, substituting150 mM gulono-γ-lactone for 150 mM pantoyl-γ-lactone. Analysis by HPLCshowed that gulonic acid was formed. The complex shape of the gulonicacid peak did not allow accurate quantification of the productformation.

In addition, ULA was assayed for the ability to catalyze the hydrolysisof pantoyl-γ-lactone or gulono-γ-lactone. No product formation wasdetectable by HPLC. The lack of measurable activity may be due to thevery low amount of soluble ULA formed after IPTG induction.

The amount of ascorbic acid secreted into the medium when inducedcultures of glo-pET30a-Rosetta (DE3) were incubated with 1 mMgulono-γ-lactone in a mannitol minimal medium was measured as describedin Example 9. After incubation for 7 hours at 30° C. and induction with0.05 mM IPTG, the glo-pET30a-Rosetta (DE3) culture secreted 110.7 nmolof ascorbic acid per mL of fermentation broth versus 14.3 nmol secretedby the control culture of pET30a-Rosetta(DE3).

Example 9 Detection of Ascorbic Acid in Biological Samples

Ascorbic acid was measured in biological samples including fermentationbroth and cell extracts using two methods. For ascorbic acidconcentrations determined immediately after sampling, a colorimetricassay that follows the reduction of ferric (Fe(III)) to ferrous (Fe(II))ions by ascorbic acid was used as described elsewhere (Zannoni et al.,Biochemical Medicine, 11:41-48 (1974)). In this assay, the production offerrous iron is measured by the formation of a 2,2′-dipyridyl-Fe(II)complex that absorbs at 525 nm. Briefly, 0.15 mL of biological samplewas mixed with 0.956 mL 2,2′-dipyridyl reagent and incubated at 25° C.for 15 minutes. After 15 minutes, the sample was centrifuged to removeany precipitate, and the absorbance was read at 525 nm. 0.956 mL of2,2′-dipyridyl reagent contained 0.056 mL ortho-phosphoric acid (85%),0.75 mL 0.5% 2,2′-dipyridyl (prepared in hot H₂O), and 0.15 mL 1% FeCl₃(in H₂O). Standards of ascorbic acid (1.0 μg to 10 μg in 0.15 mL) wererun in parallel with the biological samples for a standard curve.

Duplicate samples were stabilized for several hours or days aftersampling using a method described elsewhere (Lykkesfeldt, AnalyticalBiochemistry, 282:89-93 (2000)). This method involves acidifying with10% meta-phosphoric acid and freezing at −80° C. followed by gentlethawing and reducing with tris(2-carboxyethyl)phosphine hydrochloride atpH 6.2 just before analysis by HPLC. The pH was reduced following thetreatment with tris(2-carboxyethyl)phosphine hydrochloride to minimizethe oxidation of ascorbic acid during analysis. Briefly, the biologicalsample was mixed with an equal volume of 10% meta-phosphoric acidcontaining 2 mM EDTA and immediately frozen at −80° C. On the day ofHPLC analysis, the sample was gently thawed. 0.1 mL of 2.5 mMtris(2-carboxyethyl)phosphine hydrochloride in 0.8 M Tris-HCl (pH 9.0)was added to 0.2 mL of thawed sample. After mixing, the solution wasincubated at 25° C. for 5 minutes, and then the pH was adjusted to 4.7by the addition of 0.7 mL of 0.46 M disodium hydrogen phosphate plus0.27 M citric acid (pH 4.5). Before injection on the HPLC, all sampleswere filtered through 0.2 μm filters. The HPLC parameters were asfollows: (1) Column: Shodex Asahipak NH₂P-50 4E; (2) Eluent: A: 20 mMNaH₂PO₄+30 mM H₃PO₄ (pH 2.2) B:CH₃CN 20A/80β isocratic gradient; (3)FlowRate: 1.0 mL/min; (4) Detector: UV (254 nm; (5) Temperature:Ambient; and (6) Retention Times: Erythorbic Acid=6.3 minutes, AscorbicAcid=7.7 minutes.

Example 10 E. coli Construct and Vitamin C Production

E. coli DH10B ElectroMAX cells were purchased from Invitrogen LifeTechnologies, Inc (Carlsbad, Calif.). E. coli Rosetta(DE3) was purchasedfrom Novagen (Madison, Wis.). E. coli strain GM48 (ATCC #39099) waspurchased from American Type Culture Collection (Rockville, Md.).Electrocompetant GM48 cells were prepared by growing cultures to mid-logphase (OD₆₀₀=0.5-0.8) in LB medium and washing 3 times with equalvolumes of ice-cold 10% glycerol followed by resuspension in ice-cold10% glycerol at a ratio of 40 μL per 1 mL of original culture and rapidfreezing of 40 μL aliquots at −80° C. E. coli expression vectorspETBlue-2 and pET 11a were purchased from Novagen (Madison, Wis.).Expand DNA polymerase and the Rapid DNA Ligation Kit was purchased fromRoche Diagnostics Corp (Indianapolis, Ind.). Microbial growth mediacomponents were from Becton Dickinson Microbiology Systems (Sparks, Md.)or VWR Scientific Products (So. Plainfield, N.J.), and other reagentswere of analytical grade or the highest grade commercially available.Primers were purchased from Integrated DNA Technologies, Inc.Restriction enzymes were from New England Biolabs, Inc (Beverly, Mass.).An Eppendorf Mastercycler Gradient thermal cycler was used for PCRexperiments. UV-visible spectrometry was done using a Bio-Rad SmartSpec3000 or a Molecular Devices SpectraMAX Plus spectrophotometer(Sunnyvale, Calif.). Electroporations were performed using a Bio-RadGene Pulser II system. Automated DNA sequencing was carried by SeqWright(Houston, Tex.).

Recombinant DNA techniques for PCR, purification of DNA, ligations, andtransformations were carried out according to established procedures(e.g., Sambrook et al., Molecular Cloning (A Laboratory Manual) SecondEdition, Cold Spring Harbor Laboratory Press (1989) and Manufacturers'Technical Bulletins).

A synthetic 3 gene operon composed of: 5′-rat glo, Cryptococcusneoformans mio, and rat glr-3′ (glo_mio_glr) was constructed by thetechnique of overlap PCR as described by Ho et al. (Gene, 77(1):51-59(1989)). Briefly, this technique allowed for the fusion of 3 independentDNA sequences (glo, mio, and glr) through the use of complimentaryoligonucleotide primers and PCR to generate DNA fragments withoverlapping ends. Primers for the synthesis of the rat glo, Cr.neofeomans mio, and rat glr sequences with appropriate overlappingsequences, Ribosomal Binding Sites (RBS), and restriction sites for thepETBlue-2 vector were designed. After PCR amplification and purificationof the PCR product from 1% agarose gels, these products were combined ina second “fusion” PCR reaction in which the overlapping ends anneal.This overlap allows each strand to serve as a primer for the extensionof the complimentary strand. The addition of oligonucleotide primers forthe ends of the fused product (the forward primer for glo and thereverse primer for glr) allowed for simultaneous amplification of thefused product. Initial PCR amplification was accomplished using thecorresponding pYES2 clones as templates. After purification of the PCRproduct from 1% agarose gels and restriction digestion with NheI/PacI ofboth the PCR products and the pETBlue-2 vector, the ligation was carriedout using the Rapid DNA Ligation Kit (Roche). The ligation mix wasdesalted and then transformed into E. coli DH10B ElectroMAX cells usingthe BioRad recommended procedure for transformation of E. coli cellsusing 0.2 cm micro-electroporation cuvettes. After recovery in SOCmedium, the transformation mixtures were plated on LB plates containingampicillin (100 μg/mL),5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal; 70 μg/mL),and isopropyl-beta-D-thiogalactopyranoside (IPTG; 80 μM). Plasmid DNA ofwhite colonies picked from the LB+ampicillin+IPTG+X-gal plates wasisolated from liquid cultures (5 mL 2× YT medium+ampicillin (100 μg/mL)grown overnight at 37° C.) and purified using a Qiagen mini-prep kit.The purified plasmids were screened by restriction digestion andverified by dideoxynucleotide chain-termination DNA sequencing. Thisconstruct was designated pETBlue-3.

The sequence for the forward primer for glo with NheI site and syntheticRBS was as follows: 5′-GGCGGCTAGCGAAGGAGATATACCATGGTCCATGGGTACAAAG-3′(SEQ ID NO:49). The sequence for the reverse primer for glo with SphI,XhoI sites and synthetic RBS was as follows:5′-CTTCGGGAGCGTGCATGGTATATCTCCTTCTGCATGCTCGAGTTAGTAG-3′ (SEQ ID NO:50).The sequence for the forward primer for mio with SphI, XhoI sites andsynthetic RBS was as follows:5′-CTACTAACTCGAGCATGCAGAAGGAGATATACCATGCACGCTCCCGAAGTC-3′ (SEQ IDNO:51). The sequence for the reverse primer for mio with MluI and AscIsites and synthetic RBS was as follows:5′-GAGGCCGCCATGGTATATCTCCTTCACGCGTGGCGCGCCTACCACTGCACCTCCTCAG-3′ (SEQ IDNO:52). The sequence for the forward primer for glr with AscI and MluIsites and synthetic RBS was as follows:5′-GGTAGGCGCGCCACGCGTGAAGGAGATATACCATGGCGGCCTCCAGTGTCCT-3′ (SEQ IDNO:53). The sequence for the reverse primer for glr with PacI and XbaIsites was as follows: 5′-CGGCTTAATTAATGCGGCCCTCTAGATCAGTAT-3′ (SEQ IDNO:54). The italics indicate restriction sites, the bold letteringindicates the start and stop codons, and the underlined sequencesindicate RBS sequences.

The S. cerevisiae genes ino1 and ilr1 were amplified from S. cerevisiaegenomic DNA by PCR and inserted into pETBlue-3 as MluI/AscI fragmentsbetween the mio and glr sequences. These constructs (pETBlue-3+ino1 andpETBlue-3+itr1, respectively) were screened for correct orientation ofthe MluI/AscI fragment by MluI/AscI digestion.

pETBlue-3, pETBlue-3+ino1, and pETBlue-3+itr1 were each transformed intoelectrocompetant E. coli strain GM48 (dam methylation (−)) using theBioRad recommended procedure for transformation of E. coli cells using0.2 cm micro-electroporation cuvettes. After recovery in SOC medium, thetransformation mixtures were plated on LB plates containing ampicillin(100 μg/mL). Plasmid DNA of colonies picked from the LB+ampicillinplates was isolated from liquid cultures, purified using a Qiagenmini-prep kit, and digested with XbaI. The glo_mio_glr,glo_mio_ino1_glr, and the glo_mio_itr1_glr operons were each purifiedfrom 1% agarose gel and ligated into XbaI/Shrimp Alkaline Phosphatasedigested pET11a. The ligation was carried out using the Rapid DNALigation Kit (Roche). The ligation mix was desalted and then transformedinto E. coli DH10B ElectroMAX cells using the BioRad recommendedprocedure for transformation of E. coli cells using 0.2 cmmicro-electroporation cuvettes. After recovery in SOC medium, thetransformation mixtures were plated on LB plates containing ampicillin(100 μg/mL). Plasmid DNA of colonies picked from the LB+ampicillinplates was isolated from liquid cultures and purified using a Qiagenmini-prep kit. The purified plasmids were screened by restrictiondigestion and verified by dideoxynucleotide chain-termination DNAsequencing. The resulting constructs were designated pET11a-3,pET11a-3+ino1, and pET11a+itr1, respectively. Orientation of the operonsin the pET11a vector was confirmed by restriction digest with BamHI.

The sequence for the forward primer for ino1 with AscI and SacII sitesand synthetic RBS was as follows:5′-CGCAGGCGCGCCCCGCGGAAGGAGATATACCATGTTAGTTTTATCCTTGATTTA-3′ (SEQ IDNO:55). The sequence for the reverse primer for ino1 with MluI and ApaIsites was as follows: 5′-GCATACGCGTGGGCCCGTTACAACAATCTCTCTTCGAATCT-3′(SEQ ID NO:56). The sequence for the forward primer for itr1 with AscIand ApaI sites and synthetic RBS was as follows:5′-CGCAGGCGCGCCGGGCCCGAAGGAGATATACCATGGGAATACACATACCATA-3′ (SEQ IDNO:57). The sequence for the reverse primer for itr1 with MluI and SacIIsites was as follows: 5′-GCATACGCGTCCGCGGCCTATATATCCTCTATAATC-3′ (SEQ IDNO:58). The italics indicate restriction sites, the bold letteringindicates the start and stop codons, and the underlined sequencesindicate RBS sequences.

Transformation of the pET11a vector constructs into E. coli Rosetta(DE3)expression host was carried out according to procedures specified byNovagen and plated on LB+100 μg/mL ampicillin+34 μg/mL chloramphenicol.Colonies from each plate were picked and analyzed by plasmid isolationand gel visualization. One isolate from each construct was chosen forexpression studies.

E. coli Rosetta(DE3) cells carrying one of the described vectorconstructs (pET11a-3, pET11a-3+ino1, or pET11a-3+itr1) and the pET11aparent vector were grown in glycerol defined media containing 100 μg/mLampicillin+34 μg/mL chloramphenicol for 8 hours at 37° C. with shakingand placed at 4° C. overnight. The cells were pelleted by centrifugationat 3500×g for 10 minutes at 4° C., and the pellets were each suspendedin 5 mL glycerol defined medium containing 100 μg/mL ampicillin+34 μg/mLchloramphenicol. The OD₆₀₀ of each resuspended culture was determined,and the amount of culture necessary to obtain an OD₆₀₀ of 0.08 to 0.16in 30 mL of glycerol defined media containing 100 μg/mL ampicillin+34μg/mL chloramphenicol was calculated. The calculated volume of cells wasinoculated, and each construct was grown at 37° C. with shaking at 225rpm for 2.5 hours until the OD₆₀₀ of each culture reached ˜0.6. Three mLaliquots of fermentation broth were removed and centrifuged to removethe cells. The supernatants were diluted with an equal volume of 10%meta-phosphoric acid containing 2 mM EDTA as described in herein tostabilize the ascorbic acid in the samples. Myo-inositol (pET11a,pET11a-3, and pET11a-3+ilr1) or glucose (pET11a-3+ino1) was added to afinal concentration of 1%. 10 μM ferrous ammonium sulfate and 100 μMIPTG were added to all cultures. After 3 and 6 hours, 3 mL aliquots offermentation broth were removed and centrifuged to remove the cells. Thesupernatants were diluted with an equal volume of 10% meta-phosphoricacid to stabilize the ascorbic acid in the samples. Upon completion ofthe time course, all samples were reduced withtris(2-carboxyethyl)phosphine hydrochloride and assayed using the2,2′-dipyridyl reagent.

The concentration of ascorbic acid in the fermentation broth for eachconstruct at each time point was divided by the OD₆₀₀ of the constructat that time point. As shown in FIG. 19, ascorbic acid was produced withall constructs. These results indicate that glo, glr, and mio can beused to produce ascorbic acid.

Example 11 Yeast Construct and Vitamin C Production

E. coli DH 10B ElectroMAX cells were purchased from Invitrogen LifeTechnologies, Inc (Carlsbad, Calif.). S. cerevisiae strain YPH500 andthe pESCleu and pESCtrp vectors were obtained from Stratagene, Inc (LaJolla, Calif.). Two genes can be cloned into each of these vectors. Eachvector contains separate multiple cloning sites behind either a Gall orGal10 promoter. Expand DNA polymerase and the Rapid DNA Ligation Kit wasobtained from Roche Diagnostics Corp (Indianapolis, Ind.). Microbialgrowth media components were obtained from Becton Dickinson MicrobiologySystems (Sparks, Md.) or VWR Scientific Products (So. Plainfield, N.J.).Other reagents were of analytical grade or the highest gradecommercially available. Primers were purchased from Integrated DNATechnologies, Inc. Restriction enzymes were obtained from New EnglandBiolabs, Inc (Beverly, Mass.). Electrophoresis was carried out using aBio-Rad Protean II minigel system (protein) and a Bio-Rad Mini-Sub CellGT system (DNA) (Bio-Rad Laboratories, Hercules, Calif.). An EppendorfMastercycler Gradient thermal cycler was used for PCR experiments.UV-visible spectrometry was done using a Bio-Rad SmartSpec 3000 or aMolecular Devices SpectraMAX Plus spectrophotometer (Sunnyvale, Calif.).Electroporations were performed using a Bio-Rad Gene Pulser II system.Automated DNA sequencing was carried by SeqWright (Houston, Tex.).

Recombinant DNA techniques for PCR, purification of DNA, ligations, andtransformations were carried out according to established procedures.The sequences for GLO and/or (ULA or LHA) were cloned into a pESC-leuvector, while the sequences for GLR and MIO were cloned into a pESC-trpvector for dual transformation into the S. cerevisiae strain YPH500.Primers for the synthesis of the rat glo, rat glr, Cr. neoformans mio,Zymomonas mobilis ula, and Fusarium oxysporum lha sequences withappropriate restriction sequences for the pESC vectors 5′ of eachsequence's ATG start codon and 3′ of each sequence's stop codon weredesigned for PCR amplification using the corresponding pYES2 clones astemplate.

The sequence for the forward primer for glo with SalI site was asfollows: 5′-GGCCGTCGACCATAATGGTCCATGGGTACA-3′ (SEQ ID NO:59). Thesequence for the reverse primer for glo with XhoI site was as follows:5′-AATTCTCGAGTTAGTAGAAGACTTTCTCCAGGT-3′ (SEQ ID NO:60). The sequence forthe forward primer for glr with ApaI site was as follows:5′-GGAAGGGCCCATAATGGCGGCCTCCAGTGTCCTCCTGC-3′ (SEQ ID NO:61). Thesequence for the reverse primer for glr with HindIII site was asfollows: 5′-GGCCAAGCTTTAGATCAGTATGGGTCATTA-3′ (SEQ ID NO:62). Thesequence for the forward primer for ula with SpeI site was as follows:5′-CCGGACTAGTATAATGGCCACTGGTCGTAT-3′ (SEQ ID NO:63). The sequence forthe reverse primer for ula with PacI site was as follows:5′-GGCGTTAATTAACCCTCTAGATTACCAGAAAATAAG-3′ (SEQ ID NO:64). The sequencefor the forward primer for lha with SpeI site was as follows:5′-CGGCACTAGTATAATGGCTAAG-CTTCCTTCTACGGCTCAG-3′ (SEQ ID NO:65). Thesequence for the reverse primer for lha with PacI site was as follows:5′-GGCCTTAATTAACTAATCATA-GAGCTTGGGACCCGAAGC-3′ (SEQ ID NO:66). Thesequence for the forward primer for mio with SpeI site was as follows:5′-GGCCACTAGTATAATG-GACGCTCCCGAAGTCA-3′ (SEQ ID NO:67). The sequence forthe reverse primer for mio with PacI site was as follows:5′-GGCCTTAATTAATAGACTACCACTG-CACCTCCTCAG-3′ (SEQ ID NO:68). The italicsindicate the restriction sites, while the bold lettering indicates thestart and stop codons.

After purification of the PCR products from 1% agarose gels andrestriction digestion of both the PCR products and the pESC vectors, theligations were carried out using the Rapid DNA Ligation Kit (Roche). Theligation mixes were desalted and then transformed into E. coli DH10BElectroMAX cells using the BioRad recommended procedure fortransformation of E. coli cells using 0.2 cm micro-electroporationcuvettes. After recovery in SOC medium, the transformation mixtures wereplated on LB plates containing ampicillin at 100 μg/mL. Plasmid DNA wasisolated from liquid cultures (5 mL 2× YT medium+ampicillin (100 μg/mL)grown overnight at 37° C.) of colonies picked from the LB+ampicillinplates and purified using a Qiagen mini-prep kit. The purified plasmidswere screened by restriction digestion and verified by dideoxynucleotidechain-termination DNA sequencing.

S. cerevisiae str. YPH500 competent cells were prepared using an S.c.EasyComp™ Transformation Kit (Invitrogen Corp, Carlsbad, Calif.).Aliquots (50 μL) were frozen at −80° C. and thawed just prior to use.

Transformation of the pESC vector constructs into S. cerevisiae str.YPH500 competent cells was carried out using the S.c. EasyComp™Transformation Kit. The vector construct glo-pESCtrp, (glo+ula)-pESCleu,or (glo+lha)pESCleu was co-transformed with the (glr+mio)-pESCtrp vectorconstruct. A 100 μL aliquot from each transformation reaction was spreadon SC-leu-trp plates. The plates were incubated for 2 days at 30° C.Colonies from each plate were picked and analyzed by PCR. One isolatefrom each construct that generated the expected PCR products (evaluatedby agarose gel electrophoresis) was chosen for expression studies.

S. cerevisiae str. YPH500 cells carrying one of the described vectorconstructs or the vectors without inserts were grown in 5 mL SC-trp-leumedium containing 2% raffinose and 0.2% glucose overnight at 30° C. withshaking. The cells were pelleted by centrifugation at 1500×g for 10minutes, and the pellets were each suspended in 40 mL SC-leu-trp mediumcontaining 2% raffinose. The resulting cell suspensions were incubatedat 30° C. overnight with shaking. The OD₆₅₀ of each overnight culturewas determined, and the amount of overnight culture necessary to obtainan OD₆₅₀ of 0.2 to 0.4 in 100 mL of SC-leu-trp containing 0.2% galactose(induction medium) was calculated. The calculated volume of cells wascentrifuged at 1500×g for 10 minutes at 4° C., and the pellet wasresuspended in 2 mL of induction medium and added to 150 mL inductionmedium containing 1% myo-inositol and 0.5% raffinose. Each construct wasgrown at 30° C. with shaking at 225 rpm from 0 to 19 hours. At 0, 4, 8,and 19 hours, aliquots of fermentation broth were removed andcentrifuged to remove the cells. The supernatants were assayed forascorbic acid using the 2,2′-dipyridyl reagent.

The concentration of ascorbic acid in the vector control samples foreach time point was subtracted from the values determined for the othersamples. At 4 hours, the maximum amount of ascorbic acid was produced inthe 3 sequence construct composed of glo, glr, and mio on the pESCvectors (7.9 mg/L supernatant) and the 4 sequence construct with theadditional lha sequence (9.4 mg/L supernatant; FIG. 20). These resultsindicate that glo, glr, and mio can be used to produce ascorbic acid andthat lha can enhance this production.

Example 12 Increasing Myo-Inositol Oxygenase Activity In Vivo

An increase in the expression of mio in mammalian organs (e.g., humankidneys) is designed to lead to an increase in the production ofglucuronic acid. The increased level of glucuronic acid producedresulting from mio expression is designed to lead to the increasedproduction of glucaric acid through the use of an aldehydedehydrogenase. This increased level of glucaric acid is designed toinhibit the activity of beta-glucuronidase, thus detoxifying toxicmetabolites in the body.

Oligonucleotide primers homologous to the 5′ and 3′ ends of the humanmyo-inositol oxygenase (mio) sequence (GenBank Accession No.XM_(—)010057; gi|18594511) are designed and used to amplify human miofrom a human cDNA library by PCR. The sequence of the forward primer formio with XbaI site is as follows:5′-AATCTCTAGA-ATGAAGGTGACGGTGGGCCCAGAC-3′ (SEQ ID NO:69). The sequenceof the reverse primer for mio with KpnI site is as follows:5′-CTATGGTACCTCACCAGCTC-AGGATGCC-3′ (SEQ ID NO:70). The italicsindicates restriction sites, and the bold lettering indicates the startand stop codons.

After purification of the PCR product from 1% agarose gels andrestriction digestion with XbaI/KpnI of both the PCR products and thepSHUTTLE (Clontech) vector, the ligation is carried out using the RapidDNA Ligation Kit (Roche). The ligation mix is desalted and thentransformed into E. coli DH10B ElectroMAX cells using the BioRadrecommended procedure for transformation of E. coli cells using 0.2 cmmicro-electroporation cuvettes. After recovery in SOC medium, thetransformation mixtures are plated on LB plates containing kanamycin (50μg/mL).

Plasmid DNA of colonies picked from the LB+kanamycin is isolated fromliquid cultures (5 mL 2× YT medium+kanamycin (50 μg/mL) grown overnightat 37° C.) and purified using a Qiagen mini-prep kit. The purifiedplasmids are screened by restriction digestion and verified bydideoxynucleotide chain-termination DNA sequencing. This construct isdesignated pSHUTTLE-mio.

pSHUTTLE-mio is digested with PI-SceI and I-CeuI and purified from 1%agarose gel. The resulting fragment containing the mio sequence isisolated and ligated into predigested Adeno-X viral DNA (Clontech). Theligation is carried out using the Rapid DNA Ligation Kit (Roche). Theligation mix is desalted, digested with SwaI, desalted, and thentransformed into E. coli DH10B ElectroMAX cells using the BioRadrecommended procedure for transformation of E. coli cells using 0.2 cmmicro-electroporation cuvettes. After recovery in SOC medium, thetransformation mixtures are plated on LB plates containing kanamycin (50μg/mL). Plasmid DNA of colonies picked from the LB+kanamycin plates areisolated from liquid cultures (5 mL 2× YT medium+kanamycin (50 μg/mL)grown overnight at 37° C.) and purified using a Qiagen mini-prep kit.The purified plasmids are screened by restriction digestion and verifiedby dideoxynucleotide chain-termination DNA sequencing. The resultingconstruct is designated Adeno-X-mio.

Adeno-X-mio is transfected into low passage HEK 293 cells (Clontech),and adenoviral DNA is harvested in 4-7 days. 200 μL of Adeno-X-mio(>10¹⁰ pfu/mL) is injected into the kidneys of 3 month old Fisher rats(experimental group) and 200 μL of Adeno-X (>10¹⁰ pfu/mL) is injectedinto the kidneys of 3 month old Fisher rats (control group). Three dayslater, the rats of both the experimental and control groups aresacrificed, and the kidney tissue is assayed for mio activity as well aslevels of glucuronic acid and glucaric acid. Levels of mio activity,glucaric acid, and glucuronic acid are compared between the experimentaland control groups.

Example 13 Cloning and Expression of B. subtilis ycbD and ycbE Sequencesinto E. coli

Bacillus subtilis ATCC strain 6051 was purchased from ATCC. PfuTurbo DNApolymerase was from Stratagene (La Jolla, Calif.). Microbial growthmedia components were from Becton Dickinson Microbiology Systems(Sparks, Md.) or VWR Scientific Products (So. Plainfield, N.J.).

The ycbD (accession number 16077068; region gi|16077068:268838-270304)and ycbE (accession number 16077068; region gi|16077068:270388-271755)sequences from B. subilis were cloned into the pET30a (Novagen) andpPRONde vectors. The pPRONde vector is a derivative of the pPROLAR.A122vector (Clontech Laboratories, Inc) in which an NdeI site has beenintroduced at bp 132 by site-directed mutagenesis. The ycbD gene hasbeen identified as encoding an aldehyde dehydrogenase, while the ycbEgene has been identified as encoding a glucaric acid transporter.

Primers with compatible restriction sequences with the pET30a andpPRONde multiple cloning sites 5′ of the ycbD ATG start codon and 3′ ofthe ycbE stop codon were designed, and the sequences were amplified byPCR using B. subtilis genomic DNA as template. A mixture of 4 partsExpand DNA polymerase and 1 part PfuTurbo DNA polymerase was used in theamplification reactions. The sequence of the forward primer with NdeIsite was as follows: 5′-GCG ATT CCA TAT GTC TGT GAT CAC GGA ACA AAA CACGTA C-3′ (SEQ ID NO:71). The sequence of the reverse primer with BamHIsite was as follows: 5′-GCG CGGATC CAG GCT TAA TTA AGC TTA GAC AGG CAACGA T-3′ (SEQ ID NO:72). The italics indicate the restriction sequences,while the bold lettering indicates the start and stop codons.

Genomic DNA was purified from a culture of Bacillus subtilis ATCC strain6051 grown at 30° C. in Nutrient Broth. The Qiagen genomic tip 100/Gsystem was used to isolate the genomic DNA with the following changesfrom the manufacturer's protocol—the concentrations of proteinase K andlysozyme were doubled, and the incubation time with the enzymes was 2-3times longer.

The intervening sequence between the ycbD and ycbE sequences contains anNdeI site. The individual ycbD and ycbE sequences were purified from arestriction digest (NdeI/BamHI) of the PCR product. The ycbD sequencewas cloned into vectors pET30a and pPRONde previously digested withNdeI, while the ycbE sequence was cloned into pET30a and pPRONdepreviously digested with NdeI and BamHI. Both sequences were also clonedinto pET30a sequentially. The ycbEpET30a construct was digested withNdeI followed by ligation with the NdeI digested ycbD gene. Allrestriction digests of the PCR products and plasmids were purified from1% agarose gels. All ligations were carried out using the Rapid DNALigation Kit (Roche). The ligation mixes were desalted and transformedinto E. coli DH10B ElectroMAX cells using the BioRad recommendedprocedure for transformation of E. coli cells using 0.2 cmmicro-electroporation cuvettes. After recovery in SOC medium, thetransformation mixtures were plated on LB plates containing 50 μg/mLkanamycin. Plasmid DNA was isolated from liquid cultures (5 mL 2×YT+kanamycin (50 μg/mL)) inoculated with colonies picked from theLB+kanamycin plates and grown overnight at 37° C. The plasmid DNA waspurified using a Qiagen mini-prep kit. The purified plasmids werescreened by restriction digestion and verified by dideoxynucleotidechain-termination DNA sequencing. One silent base change was found inthe ycbD sequence compared to the GenBank sequence: C938A. One basechange was detected in the ycbE sequence compared to the GenBanksequence: C499T, corresponding to a change in the amino acid sequence ofP167S.

The pET30a constructs, verified by sequence analysis, were subclonedinto the expression host BLR(DE3) (Novagen) following the manufacturer'sprotocol. These constructs were verified by restriction digestion of thepurified plasmids.

Induction experiments with pET30a/BLR(DE3) clones were carried out in LBmedium containing 50 mg/L kanamycin. The cultures were first grown at37° C. with shaking at 225 rpm to an OD₆₅₀ between 0.5 and 0.8, andprotein expression was induced by addition of 0.1 M IPTG. The cultureswere incubated for 4 hours at 30° C. with shaking at 225 rpm andharvested by centrifugation at 21,000×g for 10 minutes. The proteinswere analyzed by SDS-PAGE on 4-15% gradient gels to check for total andsoluble protein levels at the predicted MW of the recombinant protein.Total protein samples were prepared by incubation of a cell pellet from1 mL of culture with protein loading buffer (50 mM Tris-HCl, pH 8.8, 10%glycerol, 2.0% SDS, 100 mM dithiothreitol, 0.1% bromophenol blue) for 10minutes at 95° C.

Cell extracts were prepared using Novagen BugBuster reagent (5 mLreagent per 1 g WCW) containing 1 μL of benzonase protease per mLreagent and 5 μL of Calbiochem protease inhibitor set III per mLreagent. The cell suspension was incubated at room temperature for 15minutes with gentle shaking followed by removal of the cell debris bycentrifugation at 21,000×g for 20 minutes. The supernatant (cellextract) was carefully removed and desalted by passage through aPharmacia disposable PD-10 column previously equilibrated with 25 mL of50 mM Tris-HCl, pH 8.0 containing 10 mM dithiothreitol. The proteinswere eluted using the same buffer. The desalted cell extracts were usedfor SDS-PAGE analysis of soluble proteins and for enzyme assays.

The dehydrogenase activity of the cell extracts was followed usingacetaldehyde, glucuronic acid, and glucurono-3,6-lactone as substratesin 96-well plates. Each assay mixture contained 100 mM potassiumphosphate, pH 7.8, 1 mM NADP or NAD, 200 mM substrate, and between 0.02and 0.1 mL cell extract (0.1 to 0.5 mg protein). The reactions werestarted by adding the enzyme and were incubated at 30° C. in thespectrophotometer. The dehydrogenase activity was followed by monitoringthe linear change in absorbance at 340 nm over a 10 minute time period(referenced to a reaction mixture without substrate).

The products of ycbD and ycbE were expressed in significant amounts(˜10% of total protein) in the pET30a constructs and were easilydetected in the soluble protein fractions by SDS-PAGE. NADP was thepreferred coenzyme for the dehydrogenase activity of the ycbD productfor all substrates tested. Table III summarizes the enzyme activity forthe cell extract from the [ycbd+ycde]-pET30a-BLR(DE3) construct measuredwith 0.05 mL cell extract (0.25 mg protein). Acetaldehyde was the bestsubstrate of those tested followed by glucurono-3,6-lactone, and finallyglucuronic acid. Potassium was not required for enzyme activity. When200 mM Tris-HCl, pH 7.8, was used as the buffer, the rate of oxidationof the glucurono-3,6-lactone was slightly higher than when 100 mMpotassium phosphate was used (other substrates not assayed).

TABLE III Enzymatic activities in cell extracts containing the [ycbd +ycde]-pET30a-BLR(DE3) construct. Relative Activity* Substrate NADP NADacetaldehyde 100.0 56.9 D-glucuronate 2.1 2.4 D-glucurono-3,6-lactone50.6 2.1 *activity defined as percentage of maximum activity withacetaldehyde as the substrate and NADP as the coenzyme.

Example 14 Detecting Glucaric Acid in Biological Samples

The formation of glucaric acid in biological samples (both fermentationbroth and cell extracts) is followed by HPLC. Two lactones,glucarate-1,4-lactone and glucarate-3,6-lactone, are in equilibrium withglucaric acid at neutral pH, and their formation is monitored asdescribed elsewhere (Horton and Walaszek, Carbohydrate Research,105:95-109 (1982)). Aliquots of the samples are sterile filtered through0.2 μm filters before injection on the chromatography column. The HPLCparameters are as follows: (1) Column: Aminex APX-87H 300×7.0 mm BioRad;(2) Eluent: 0.005N H₂SO₄; (3) Flow Rate: 0.6 mL/min; (4) Detector:Refractive Index; (5) Temperature: 45° C.; (6) Retention Times:Glucaric-1,4-lactone=9.0 minutes, Glucaric-3,6-lactone=9.1 minutes,Glucaric acid=8.0 minutes, Glucurono-3,6-lactone=10.8 minutes, andGlucuronic acid=8.1 minutes.

Example 15 Enzymatic Formation of Glucaric Acid from Glucuronic Acid

Glucaric acid is synthesized from glucuronic acid in a reactioncatalyzed by an enzyme with non-specific hexose oxidase activity (EC1.1.3.5). The enzyme isolated from the red alga Chondrus crispus(AAB49376.1 GI:1877522) catalyzes this reaction at 2% the rate of theoxidation of glucose. Homologs of this enzyme with fasta probabilityscores (P-scores) greater than 4e-60 were identified in Yersinia pestis(NP_(—)403 959.1; gi:16120646), Yersinia pseudotuberculosis (SangreCentre, gene sequence on Contig1834 (length 8,117), from 5,184 to3,130), Ralstonia solanacearum (NP_(—)518171.1; gi:17544769), andBurkholderia pseudomallei (Sangre Centre, gene sequence on Contig01233(length 58,761), from 23,445 to 25,103) by BLAST analysis of the aminoacid sequences.

The amount of glucaric acid formed by reaction of glucuronic acid withhexose oxidase is measured as described elsewhere (Sullivan and Ikawa,Biochimica et Biophysica Acta, 309:11-22 (1973) and U.S. Pat. No.6,251,626). In this assay, the hydrogen peroxide formed during theoxidation of glucuronic acid in the presence of peroxidase reacts with achromogenic substance, ortho-dianisidine to form a dye that absorbs at402 nm.

The assay mixture consists of enzyme sample and 0.85 mL of an assaysolution containing 0.370 mL of 0.1 M sodium phosphate buffer, pH 7.0;0.462 mL of D-glucuronic acid (varying concentrations) in 0.1M sodiumphosphate buffer, pH 7.0; 0.009 mL of horseradish peroxidase (SigmaChemicals, cat. no. P6782 or Boehringer Mannheim, cat. no. 814 393), 0.1mg/mL in water; and 0.009 mL of ortho-dianisidine-2HCl(3,3′-dimethoxybenzidine, Sigma Chemicals), 3.0 mg/mL in water. Afterincubation at room temperature for 15 to 30 minutes, the assay isstopped by addition of one drop of 37% HCl. Samples of 0.100 mL aretransferred from the assay tubes to the wells of a microtiter plate, andthe absorbance at 410 nm is read using a Molecular Devices SpectraMAXPlus spectrophotometer (Sunnyvale, Calif.). One enzyme unit is definedas the amount of enzyme that catalyzes the production of 1 nmolehydrogen peroxide per min at 25° C., pH 6.3, at a substrateconcentration of 0.05 M.

Example 16 Chemical Conversion of Glucuronic Acid to Glucaric Acid

The following method was used to convert glucuronic acid into glucaricacid. 5% Pd on carbon catalyst (10 g; 5% Pd/C catalyst; Johnson MattheyInc., Ward Hill, Mass.) was placed in a 3-neck flask with 50 mL ofdistilled water. Oxygen was bubbled through the mixture for about 15minutes. Meanwhile, 5 g of glucuronic acid was dissolved in 25 mL ofdistilled water, and the pH was adjusted to 8 by the addition of 10%sodium hydroxide. The glucuronate solution was then added to the flaskcontaining the catalyst. The flask was placed in an oil bath at 50° C.and equipped with a dropping funnel containing 10% sodium hydroxide, apH electrode, and an oxygen line with a metal frit. The reaction mixturewas stirred at 50° C., while continuously bubbling oxygen through themixture and monitoring the pH. The 10% sodium hydroxide was addedperiodically to maintain the pH above 8. After 10 hours, the catalystwas filtered off, and the reaction mixture analyzed by HPLC.

FIG. 21A depicts the LC-MS chromatograms of the starting material,glucuronic acid, the product, glucaric acid, and the reaction mixture at10 hours. FIG. 21B depicts the corresponding mass spectra. The reactionyield was determined to be greater than 90% by comparison of theionization fragments at 146.1 (major daughter fragment of glucaric acid)and 140.4 (major daughter fragment of glucuronic acid) in the massspectrum of the reaction mixture. These results demonstrate thatchemically- or enzymatically-derived glucuronic acid can be converted inhigh yield to glucaric acid, by catalytic oxidation.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated cell comprising myo-inositol and one or more exogenousnucleic acid molecules, wherein said one or more exognous nucleic acidmolecules encodes at least one polypeptide having myo-inositol oxygenaseactivity, and wherein said cell expresses said at least one polypeptide.2. The cell of claim 1, wherein said cell is a prokaryotic cell.
 3. Thecell of claim 2, wherein said prokaryotic cell is selected from thegroup consisting of Pseudomonas, Escherichia, Bacillus, Lactobacillus,Lactococcus, and Corynebacterium cells.
 4. The cell of claim 1, whereinsaid cell is a eukaryotic cell.
 5. The cell of claim 4, wherein saideukaryotic cell is selected from the group consisting of yeast, fungi,insect, and mammalian cells.
 6. The cell of claim 1, wherein said cellis selected from the group consisting of Saccharomyces, Pichia,Aspergillus, Cryptococcus, Schwanniomyces, Schizosaccharomyces,Spodoptera, Cricetulus, and Homo sapiens cells.
 7. The cell of claim 1,wherein said cell is a plant cell.
 8. The cell of claim 1, wherein saidat least one polypeptide comprises an amino acid sequence at least about70 percent identical to the sequence set forth in SEQ ID NO:12, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or
 35. 9. Thecell of claim 1, said cell further comprising a second polypeptideencoded by said one or more exogenous nucleic acid molecules.
 10. Thecell of claim 9, wherein said second polypeptide comprises glucuronatereductase activity.
 11. The cell of claim 10, wherein said secondpolypeptide comprises an amino acid sequence at least about 50 percentidentical to the amino acid sequence set forth in SEQ ID NO:36.
 12. Thecell of claim 9, wherein said second polypeptide comprises 1,4-lactonehydroxyacylhydrolase activity, D-glucono-1,5-lactone lactonohydrolaseactivity, hexose oxidase activity, or uronolactonase activity.
 13. Thecell of claim 12, wherein said second polypeptide comprises an aminoacid sequence at least about 50 percent identical to the amino acidsequence set forth in SEQ ID NO:37 or
 38. 14. The cell of claim 9,wherein said second polypeptide comprises gulono-γ-lactone oxidaseactivity, galactono-γ-lactone oxidase activity, or gulono-γ-lactonedehydrogenase activity.
 15. The cell of claim 14, wherein said secondpolypeptide comprises an amino acid sequence at least about 50 percentidentical to the amino acid sequence set forth in SEQ ID NO:39 or 40.16. The cell of claim 9, wherein said second polypeptide comprisesphosphatase activity.
 17. The cell of claim 16, wherein said secondpolypeptide comprises an amino acid sequence at least about 50 percentidentical to the amino acid sequence set forth in SEQ ID NO:41 or 44.18. The cell of claim 9, wherein said second polypeptide comprisesphytase activity.
 19. The cell of claim 18, wherein said secondpolypeptide comprises an amino acid sequence at least about 50 percentidentical to the amino acid sequence set forth in SEQ ID NO:42 or 43.20. The cell of claim 1, said cell further comprising a secondpolypeptide and a third polypeptide encoded by said one or moreexogenous nucleic acid molecules.
 21. The cell of claim 20, wherein saidsecond polypeptide comprises an activity selected from the groupconsisting of glucuronate reductase, 1,4-lactone hydroxyacylhydrolase,D-glucono-1,5-lactone lactonohydrolase, gulono-γ-lactone oxidase,gulono-γ-lactone dehydrogenase, uronolactonase, galactono-γ-lactoneoxidase, pyridine nucleotide transhydrogenase, phytase, and phosphataseactivities.
 22. The cell of claim 20, wherein said third polypeptidecomprises an activity selected from the group consisting of glucuronatereductase, 1,4-lactone hydroxyacylhydrolase, D-glucono-1,5-lactonelactonohydrolase, gulono-γ-lactone oxidase, gulono-γ-lactonedehydrogenase, uronolactonase, galactono-γ-lactone oxidase, pyridinenucleotide transhydrogenase, phytase, and phosphatase activities. 23.The cell of claim 1, wherein said cell lacks L-gulonate 3-dehydrogenaseactivity.
 24. The cell of claim 1, wherein said cell comprises a geneticmodification that reduces L-gulonate 3-dehydrogenase activity.
 25. Thecell of claim 24, wherein said genetic modification comprises a nucleicacid deletion in the genome of said cell.
 26. The cell of claim 1,wherein said cell produces ascorbic acid, glucaronic acid, or glucaricacid.
 27. The cell of claim 26, wherein said cell comprises pyridinenucleotide transhydrogenase activity.
 28. The cell of claim 1, whereinsaid cell comprises myo-inositol oxygenase activity with a specificactivity greater than 40 mg glucuronic acid per gram dry cell weight perhour.
 29. The cell of claim 1, wherein said cell comprises myo-inositoloxygenase activity such that an extract from 1×06 cells comprises aspecific activity greater than 150 μg glucuronic acid formed per 10 mgtotal protein per 10 minutes, wherein each of said 1×10⁶ cells is saidcell or a progeny of said cell.
 30. The cell of claim 1, wherein saidexogenous nucleic acid molecule comprises a promoter that is lactoseunresponsive.
 31. The cell of claim 1, wherein said polypeptide lacks anN-terminal polyhistidine tag.
 32. The cell of claim 1, wherein saidpolypeptide lacks glutathione-5-transferase sequence.
 33. The cell ofclaim 1, wherein said exogenous nucleic acid molecule is integrated intothe genome of said cell.
 34. A cell comprising a genetic modificationthat reduces myo-inositol oxygenase activity.
 35. The cell of claim 34,wherein said cell is a eukaryotic cell.
 36. The cell of claim 34,wherein said cell is a plant cell.
 37. The cell of claim 34, whereinsaid genetic modification comprises a nucleic acid deletion in thegenome of said cell.
 38. The cell of claim 34, wherein said cell lackssaid myo-inositol oxygenase activity.
 39. A cell comprising a geneticmodification that reduces L-gulonate 3-dehydrogenase activity.
 40. Thecell of claim 39, wherein said cell is a eukaryotic cell.
 41. The cellof claim 39, wherein said genetic modification comprises a nucleic aciddeletion in the genome of said cell.
 42. The cell of claim 39, whereinsaid cell lacks said L-gulonate 3-dehydrogenase activity.
 43. Anisolated nucleic acid molecule comprising a nucleic acid sequence atleast about 50 percent identical to the sequence set forth in SEQ ID NO:1 and wherein the polypeptide comprises myo-inositol oxygenase activity.44. The isolated nucleic acid molecule of claim 43, wherein said nucleicacid sequence is as set forth in SEQ ID NO:
 1. 45. An isolated nucleicacid molecule that encodes a polypeptide having an amino acid sequenceat least about 50 percent identical to the sequence set forth in SEQ IDNO: 19 and wherein the polypeptide comprises myo-inositol oxygenaseactivity.
 46. The isolated nucleic acid molecule of claim 45, whereinsaid amino acid sequence comprises SEQ ID NO: 19.